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Biochemical and molecular studies on Phenylalanine ammonia lyase in the phytopathogenic fungus Ustilago… Kim, Seong Hwan 1997

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BIOCHEMICAL AND MOLECULAR STUDIES ON PHENYLALANINE AMMONIA LYASE IN THE PHYTOPATHOGENIC FUNGUS USTILAGO MAYDIS by SEONG HWAN KIM B.Sc, Korea University, 1986 M.Sc, Korea University, 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i in THE FACULTY OF GRADUATE STUDIES (Biotechnology Laboratory/Department of Plant Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April, 1997 © Seong Hwan Kim, 1997 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the. University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date Aj>rrJ;3o,.'.ff99 !-DE-6 (2/88). ABSTRACT Phenylalanine ammonia-lyase (PAL) is the entrypoint enzyme into phenylpropanoid metabolism in plants. Little is known about PAL in fungi. In order to explore the role PAL plays in the growth and survival of fungi, the structure and regulation of fungal PAL were investigated in the phytopathogen Ustilago maydis, the causal agent of corn smut. PAL was purified from liquid-cultured cells of U. maydis using ion-exchange and gel filtration chromatography, and preparative PAGE. Its native molecular mass was estimated as 320 kDa and its subunit molecular mass was 80 kDa. No isoforms of the enzyme were detected, and there was no evidence of glycosylation of the protein. The enzyme was most active at pH 8.8-9.2 and 30°C and had a K m for L-phenylalanine of 1.05 mM. The enzyme did not deaminate L-tyrosine. The synthetic PAL inhibitor 2-aminoindan-2-phosphonic acid (AIP) strongly inhibited the enzyme, as did sulfhydryl reagents and carbonyl reagents, whereas f-cinnamic acid was only moderately inhibitory. Ustilago PAL activity had no requirement for metal ion cofactors, but was inhibited by heavy metal ions (Ag+, Cu2+, and Hg2+). Polyclonal antibodies were raised against the purified PAL protein. Using degenerate oligonucleotide primers and polymerase chain reaction, a PAL clone was isolated from a U. maydis genomic library and 3047 bp of its nucleotide sequence was determined. It contained 495 bp of 5' untranslated sequence, a 2172 bp open reading frame encoding 724 amino acids, and 380 bp of 3' untranslated sequence. No introns in the PAL-encoding gene were detected. In U. maydis, PAL was shown to be ii encoded by a single gene. This is the first work on the structure of a PAL gene from a pathogenic fungus. Substantial differences in PAL gene sequence and organization were found compared to PAL genes of other species. U. maydis PAL showed low amino acid sequence identity with other PALs (23-26% with plant PALs, 39-40% with yeast PALs). The level of amino acid identity (25%) with bacterial histidine ammonia-lyase (HAL) suggests a possible relatedness between U. maydis PAL and bacterial HAL. Western blot and immunological enzyme inhibition assay confirmed that PAL and HAL enzymes are immunologically related. Overall, protein sequence analysis suggests that modern PAL and HAL genes share a common ammonia-lyase ancestor. In U. maydis, PAL is constitutively produced at a low level but its regulation can be influenced by aromatic amino acids. L-tryptophan apparently induced the lyase enzyme. The inducibilty of PAL by L-tryptophan was also demonstrated in six other U. maydis strains and three Ustilago species tested. The enzyme is most readily induced during the early stationary phase of growth and the induced activity remains relatively constant during stationary stage. PAL induction was repressed by glucose but not by its reaction product r-cinnamic acid. Induction did not require de novo protein synthesis, suggesting that some form of post-translational protein modification or a metabolic effect may be the basis of the induction of Ustilago PAL by L-tryptophan. PAL was detected only in cell extracts and not in the growth medium. A putative biosynthetic pathway of Ustilago melanin was deduced from the examination of the metabolic fate of L-phenylalanine. Overall, the pattern of regulation of PAL induction in U. maydis was very different from patterns known for plants and other fungi. iii These results, together with evidence for genetic divergence, are consistent with a unique role for PAL in U. maydis. It remains to be determined whether this role is essential for survival and pathogenicity of this plant pathogen. iv TABLE OF CONTENTS Abstract " Table Of Contents v List Of Tables viii List Of Figures ix List Of Abbreviations xiii Acknowledgments xvi CHAPTER 1 - GENERAL INTRODUCTION 1 1.1 Phenylalanine metabolism 1 1.1.1 The biosynthesis of L-phenylalanine 1 1.1.2 The metabolic fate of L-phenylalanine 3 1.1.2.1 In mammals 3 1.1.2.2 In plants 8 1.1.2.3 In microorganisms 10 1.2 Phenylalanine ammonia-lyase (PAL) 12 1.2.1 Introduction 12 1.2.2 Commercial and medical potential of PAL 14 1.2.3 Active site and enzyme mechanism 15 1.2.4 Structural properties 16 1.2.4.1 PAL proteins 16 1.2.4.2 PAL-encoding genes 19 1.2.4.3 PAL promoter 19 1.2.5 Functional properties 20 1.2.6 Regulation 22 1.2.6.1 Factors influencing on PAL levels 22 1.2.6.2 Mechanisms for the regulation of PAL activity ... 23 1.2.6.3 Regulation of PAL gene expression 24 1.2.6.4 Co-ordinate regulation 27 1.3 Ustilago maydis 28 1.3.1 Corn smut disease 28 1.3.2 The life cycle and mating of U. maydis 29 1.3.3 U. maydis as a model fungus 31 1.4 Rationale and Objectives 33 CHAPTER 2 - PURIFICATION AND CHARACTERIZATION OF PHENYLALANINE AMMONIA-LYASE FROM USTILAGO MAYDIS 35 2.1 Introduction 35 2.2 Materials and methods 36 2.2.1 Materials 36 2.2.2 Fungal cultures 37 2.2.3 Purification of U. maydis phenylalanine ammonia-lyase .... 37 2.2.4 Enzyme assay 38 V 2.2.5 Polyacrylamide gel electrophoresis 39 2.2.6 Antibody production and immuno-affinity chromatography . 40 2.2.7 Western blot 40 2.3 Results 41 2.3.1 Purification of PAL 41 2.3.2 Antibody production and characterization 47 2.3.3 Western blotting 49 2.3.4 Stability 49 2.3.5 pH optimum 53 2.3.6 Catalytic properties 53 2.3.7 Inhibitors and activators 53 2.4 Discussion 59 CHAPTER 3 - CLONING AND CHARACTERIZATION OF THE PAL GENE FROM USTILAGO MAYDIS: RELATIONSHIPS WITH OTHER PHENYLALANINE AMMONIA-LYASES AND WITH BACTERIAL HISTIDINE AMMONIA-LYASES 62 3.1 Introduction 62 3.2 Materials and methods 66 3.2.1 Microorganisms and media 66 3.2.2 Materials 66 3.2.3 Nucleic acid manipulation 68 3.2.4 cDNA library screening 68 3.2.5 Design of oligonucleotide primers 69 3.2.6 PCR amplification of a putative U. maydis PAL gene fragment 69 3.2.7 Cloning of putative U. maydis PAL PCR product 71 3.2.8 DNA sequencing 72 3.2.9 Sequence comparison and analysis 72 3.2.10 Genomic-cosmid library screening 73 3.2.11 Southern hybridization 74 3.2.12 Total RNA isolation and Northern blot hybridization 75 3.2.13 N-terminal and internal peptide sequencing of U. maydis PAL 75 3.2.14 Enzymes and antibodies 76 3.2.15 Western blot and enzyme inhibition 76 3.2.16 Assay of enzyme activity 77 3.3 Results 77 3.3.1 PCR amplification of a U. maydis PAL sequence from genomic DNA 77 3.3.2 Isolation and sequencing of PAL genomic-cosmid clones 78 3.3.3 Nucleotide sequence and deduced amino acid sequence of U. maydis PAL 83 3.3.4 Genomic organization of PAL in U. maydis 88 3.3.5 Northern blot analysis of PAL transcript 88 vi 3.3.6 Protein sequence comparison 95 3.3.7 Immunological relationships 97 3.4 Discussion 110 CHAPTER 4 - REGULATION OF PHENYLALANINE AMMONIA-LYASE BY L-TRYPTOPHAN IN USTILAGO MAYDIS 118 4.1 Introduction 118 4.2 Materials and methods 120 4.2.1 Fungal strains and cultures 120 4.2.2 Chemicals 122 4.2.3 Enzyme extraction and assay 122 4.2.4 Immuno-blot analysis 123 4.2.5 Immunoprecipitation of radiolabeled PAL protein 123 4.2.6 Phenylalanine catabolism analysis 124 4.3 Results 125 4.3.1 Induction experiments 125 4.3.2 PAL induction period 128 4.3.3 PAL inducibility by tryptophan isomers, precursors, and metabolites 130 4.3.4 Tryptophan effect on PAL induction in other U. maydis strains and Ustilago species 134 4.3.5 Effect of carbon and nitrogen sources on PAL activity induction 134 4.3.6 Effect of inhibitors and cAMP on PAL activity in vivo 142 4.3.7 Influence of physical environments 142 4.3.8 No evidence for de novo synthesis in PAL induction 145 4.3.9 Metabolic fate of L-phenylalanine in U. maydis cultures .... 149 4.4 Discussion 152 4.4.1 PAL induction by L-tryptophan 152 4.4.2 Regulatory features of PAL 153 4.4.3 Metabolic features of phenylalanine catabolism in the context of the host-pathogen interaction 156 4.4.4 Potential roles of PAL in U. maydis 157 CHAPTER 5 - CONCLUSIONS AND FUTURE DIRECTIONS 160 5.1 Conclusions 160 5.2 Future directions 162 Bibliography 165 Appendix A: Cloning and sequencing of a cDNA encoding aspartate semialdehyde dehydrogenase in Ustilago maydis 185 Appendix B: A partial cDNA sequence encoding the Ustilago maydis catalase-peroxidase 193 vii LISTS OF TABLES Table No. Table title Page Table 2.1. Purification of PAL from U. maydis cells 42 Table 2.2. Effect of chemical modification reagents and substrate analogues on U. maydis PAL activity 56 Table 2.3. Effect of metal ions and chelators on U. maydis PAL activity 57 Table 3.1. Escherichia coli strains used in this study 67 Table 3.2. PCR primer combinations tested for possible amplification of PAL DNA fragments from U. maydis and predicted size of the amplified products 79 Table 3.3. BLAST search results with the deduced U. maydis PAL protein sequence 86 Table 3.4. Deduction of potentially biologically significant sites in the protein sequence (724 aa) of U. maydis PAL 89 Table 3.5. Codon usage in the U. maydis PAL gene 90 Table 3.6. Inferred amino acid composition of U. maydis PAL 91 Table 3.7. Comparison of protein sequence identity and similarity of phenylalanine ammonia-lyase (PAL) and histidine ammonia-lyase (HAL) among different organisms 96 Table 3.8. Inhibition of PAL and HAL activity by antisera raised against the two ammonia-lyases 108 Table 4.1. Ustilago species and U. maydis strains used in the study 121 Table 4.2. Effect of tryptophan analogs and tryptophan-related metabolites on PAL induction in U. maydis 133 Table 4.3. Effect of actinomycin D and cycloheximide on U. maydis PAL activity 146 Table 4.4. PAL protein synthesis in L-tryptophan-induced and -uninduced conditions 147 Appendix B Table 1. BLAST search results with the protein sequence deduced from the U. maydis 1725-bp cDNA in Fig. 1 196 viii LIST OF FIGURES Figure No. Figure title Page Figure 1.1. The pathway from erythrose 4-phosphate to chorismic acid 2 Figure 1.2. The pathway from chorismic acid to aromatic amino acids 4 Figure 1.3 The conversion of phenylalanine to tyrosine and other metabolites 6 Figure 1.4. The conversion of tyrosine to catecholamines 7 Figure 1.5. Schematic representation of general phenylpropanoid metabolism 9 Figure 1.6. Deamination of aromatic amino acids by ammonia-lyases 13 Figure 1.7. Schematic representation of the life cycle of Ustilago maydis 30 Figure 2.1. Elution profiles of U. maydis PAL and protein after DEAE cellulose chromatography (upper) and Bio-Gel A-0.5m gel filtration (lower) 43 Figure 2.2. Native PAGE analysis of purified PAL from U. maydis 44 Figure 2.3. Native molecular weight determination of U. maydis PAL by non-denaturing PAGE 45 Figure 2.4. Isoelectric focusing PAGE (A) and SDS-PAGE (B) analysis of U. maydis PAL enzyme 46 Figure 2.5. Inhibition of U. maydis PAL activity by U. maydis PAL antibodies 48 Figure 2.6. Western blot analysis of reciprocal cross-reactivity between recombinant poplar PAL, U. maydis PAL and their respective antisera 50 Figure 2.7. Western blot analysis of U. maydis PAL subunit sizes at different stages of enzyme purification 51 Figure 2.8. Effect of temperature on U. maydis PAL activity 52 Figure 2.9. Effect of pH on U. maydis PAL activity 54 Figure 2.10. Kinetic analysis of partially purified U. maydis PAL 55 ix Figure 3.1. Schematic diagram indicating generic structure of a PAL gene and locations of primers (=>) used in PCR to amplify parts of the U. maydis PAL gene 70 Figure 3.2. PCR amplification of putative PAL DNA fragment from U. maydis genomic DNA using various PAL primer combinations 80 Figure 3.3. PCR screening of U. maydis cosmid library pools using UMPAL3 and UMPAL2 primers 81 Figure 3. 4. Strategy employed in the nucleotide sequencing of the U. maydis PAL genomic-cosmid clone 82 Figure 3.5. Complete nucleotide sequence of U. maydis PAL gene 85 Figure 3.6. Deduced amino acid sequence of U. maydis PAL 87 Figure 3.7. Hydropathy profile of U. maydis PAL from amino acid 1 to amino acid 724 92 Figure 3.8. Southern blot analysis of the U. maydis PAL gene 93 Figure 3.9. Northern blot analysis of PAL transcript 94 Figure 3.10. Comparison of the deduced amino acid sequence of the U. maydis PAL with the deduced amino acid sequences of PAL and HAL from various organisms 103 Figure 3.11. Dendrogram of the inferred protein sequences of PALs and HALs from various organisms using PC-GENE Clustal Program 104 Figure 3.12. Native page (top) and western blots (center and bottom) of fungal PAL, plant PAL and bacterial HAL using polyclonal antibodies raised against U. maydis PAL (A), popular PAL (B), PAL (C), P. fluorescens HAL (D), and P. putida HAL (E) 105 Figure 3.13. Hydrophilicity profiles of PALs and HALs from different organisms 109 Figure 4.1. U. maydis cell growth and PAL activity in different media 126 Figure 4.2A. Effect of aromatic amino acids on PAL induction in U. maydis 127 X Figure 4.2B. Cell growth and PAL activity of U. maydis grown on tryptophan or phenylalanine as sole carbon and nitrogen sources 127 Figure 4.3. Induction of U. maydis PAL at different concentrations of L-tryptophan 129 Figure 4.4. Effect of tryptophan addition on cell growth and PAL induction in U. maydis 131 Figure 4.5. Comparison of PAL inducibility in U. maydis by different optical isomers of tryptophan 132 Figure 4.6. Effect of L-tryptophan supplement on PAL induction in different U. maydis strains 135 Figure 4.7. Effect of L-tryptophan supplement on PAL induction in different Ustilago species 136 Figure 4.8. Effect of various carbon sources on U. maydis cell growth and PAL activity 137 Figure 4.9A. Effect of glucose on U. maydis PAL induction by L-tryptophan 139 Figure 4.9B. Effect of time of glucose addition on U. maydis PAL induction 139 Figure 4.10. Effect of nitrogen source on U. maydis cell growth and PAL activity 140 Figure 4.11. Effect of the ratio of NH4++:N03" on PAL activity in U. maydis 141 Figure 4.12. Effect of cAMP (1mM), AIP (100|AM), and f-cinnamic acid (0.3mM)on cell growth and PAL activity of U. maydis 143 Figure 4.13. Effect of L-tryptophan supplement on PAL induction in the U. maydis adenylate cyclase-deficient (uad) and protein kinase A-deficient (ubd, adrl) mutants 144 Figure 4.14. Western blot analysis of PAL production in U. maydis growth in L-tryptophan-induced and-uninduced conditions 148 xi Figure 4.15. Autoradiogram of the radioactive metabolites recovered from U. maydis growing in L-[U-14C]-phenylalanine or 150 [U-14C]-f-cinnamic acid Figure 4.16. Schematic diagram of catabolic pathway for phenylalanine in U. maydis  1 5 1 Appendix A Figure 1. The nucleotide sequence and deduced amino acid sequence (upper case) of the U. maydis ASADH cDNA (GenBank accession No. D11111) 186 Figure 2. Southern blot analysis of the U. maydis ASADH gene 188 Figure 3. PCR amplification and Southern blot analysis of ASADH gene from U. maydis genomic DNA and ASADH cDNA 189 Figure 4. Conserved regions in ASADH-encoded amino acid sequences from Um (U. maydis), Bs (Bacillus subtilus), Cg (Corynebacterium glutamicum), Li (Leptospira interogans), Sm (Streptococcus mutans), Sc (Saccharomyces cerevisiae), and Vc (Vibrio cholerae) 190 Appendix B Figure 1. Nucleotide sequence of a 1725-bp cDNA contained in a positively selected cDNA clone during the immunoscreening of U. maydis PAL cDNA clone (chapter 3) 195 xii LIST OF ABBREVIATIONS 5', 3' denotes 5'-hydroxy or 3'-phosphate end of sequence aa amino acid A, C. G, T nucleotides adenosine, cytosine, guanosine, thymidine AIP 2-aminoindan-2-phosphonic acid AOPP L-a-aminooxy-p-phenylpropionic acid ASADH aspartate semialdehyde dehydrogenase bp base pair BSA bovine serum albumin cAMP adenosine-3', 5-monophosphate cDNA complementary deoxyribonucleic acid C4H cinnamic acid 4-hydroxylase (EC1.14.13.11) CHI chalcone isomerase CHS chalcone synthase (EC 2.3.1.74) Ci Curie 4CL 4-coumarate:CoA ligase (EC6.2.1.12) CAPS 3-(cyclohexylamino)-1-propanesulphonic acid Da Dalton DMSO dimethyl sulphoxide DTT dithiothreitol EC enzyme classification EDTA ethylenediaminetetra-acetic acid GUS p-glucuronidase hr hour(s) xiii IEF isoelectric-focussing kb kilobase kDa kilodalton Km, the Michaelis-Menten constant AZAPII lambda ZAPII phagemid vector (Stratagene) u, micro m milli M molar Mr relative molecular mass min minute(s) mRNA messenger ribonucleic acid MW molecular weight No. number nt nucleotide O.D. optical density ORF open reading frame PAGE polyacrylamide gel electrophoresis PAL phenylalanine ammonia-lyase (EC 4.3.1.5) PBS phosphate buffered saline p para PCR polymerase chain reaction PDA potato dextrose agar PEG polyethylene glycol Phe phenylalanine xiv pi isoelectric point PMSF phenylmethanesulphonyl fluoride PVDF polyvinylidene difluoride P V P polyvinyl-pyrrolidone RT room temperature S.D. standard deviation SDS sodium dodecyl sulfate sp species t trans TCA trichloroacetic acid TLC thin layer chromatography Tris tris-(hydroxymethyl)-aminoethane Trp tryptophan Tyr tyrosine UV ultraviolet V m a x maximum velocity Vol volume w/w weight by weight w/v weight by volume XV ACKNOWLEDGMENTS It is a great pleasure to acknowledge the experienced and excellent guidance of my supervisor, Dr. Brian Ellis for his advice, encouragement, dedication throughout my Ph.D. program from beginning to end. He has also given me the opportunity to present parts of the results of this thesis work at various conferences of scientific societies such as The Phytochemical Society of North America, The Canadian Phytopatholoical Society, International Mycological Congress, and The International Society of Plant Molecular Biology. I valued Brian's enthusiasm and interest in the work and science, and his willingness to generously give of his time and to share of his years of experience. Without his help, this thesis shouldn't be completed. I would like to express my appreciation to my advisory committee members Dr. Bob Copeman, Dr. Jim Kronstad, and Dr. Helene Sanfacon for their insightful suggestion, invaluable instruction, and sound advice. Special thanks are expressed to Dr. Jim Kronstad for his helpful ideas, sharing Lab facilities, and providing all the Ustilgo fungal cultures used in this study. I wish to thank Dr. Richard Dixon (Samuel Robert Noble Foundation, USA) for his gift of alfalfa PAL antiserum, Dr. J. Zon (Technical University Wroclaw, Poland) for his gift of AIP, Dr. Allen Phillips (Pennsylvania State University, USA) for his gift of Psedomonas HAL antiserum, Dr. Robin Taylor (University of Toronto) for his gift of rat HAL cDNA clone, and Dr. Andre Levesque (Pacific Agri-Food Research Centre, Summerland), for the use of an automatic DNA Sequencer for the DNA sequencing of Ustilago PAL gene. Thanks are also extended to Dr. Neil Towers (Botany Department, UBC) for his concern, interest, and suggestions, and Prof. Tae-Ju Choi (Botany Department, UBC) for his encouragement and for providing yeast cultures. Appreciation is expressed to the former and the present members of Plant Biochemistry and Plant Molecular Biology Lab Group (including Monica Lam, Leroy Scrubb, Shawn Wang, Grant McKegney, Amrita Singh, Dr. Aviva Pri-Hadash, Jennifer Norton, Stefanie Butland, Jason Galpin, Kim MacDonald, Bjorn Orvar, Marcus Samuel and Pat Harrison), Fungal Molecular Genetics Lab Group (Dr. Guss Bakkeren, Dr. Scott Gold, Dr Luc Giasson, Dr. Franz Durrenberger, Arthur Lee, Katherine Barrett, Barbara Kukan), and the former members of Phenylalanine Ammonia-Lyase Discussion Club (including Dr. Carl Douglas, Dr. Beth Molitor) for their assistance, understanding, sharing ideas and techniques, valuable discussion, and fruitful relationship. I am indebted to Plant Science Staff (Robyne Allan, Bev Busch, Ashley Herath, and Derek White) for their kindness, generosity, and support. I am also grateful to other graduate colleagues of Department of Plant Science, UBC, for their kind and constructive assistance in conducting my Ph.D. program. xvi Gratitude is due to the Natural Sciences and Engineering Research Council of Canada for financial support, and the UBC authorities for providing a University Graduate Fellowship (UBC). Special gratitude is extended to Stefanie Butland for the contribution of her time and resources with patience and kindness throughout my Ph. D. study, including critical reading of this thesis, and to Dong Suk Park (National Agricultural Science and Technology Institute, Korea) for assistance during the immunoscreening work. My gratitude is also sent to the congregations of Korean Presbyterian Deung-Dae Church for their love, encouragement, and prayer for me. Sincere appreciation is given to my parents, brother, sister, and parents-in-law for their invaluable assistance, encouragement, and perseverance. Most of all, my heartfelt thank and gratitude are expressed to my wife Ji-Hwan, son Hyun Woo, and daughter Yoo Min for their endless love, patience, and understanding. Finally, I am deeply thankful to God who helps me in various ways. xvii CHAPTER ONE General Introduction 1.1 PHENYLALANINE METABOLISM 1.1.1 The biosynthesis of L-phenylalanine L-phenylalanine is one of the essential amino acids which cannot be synthesized in mammals in adequate amounts to meet the requirements for protein synthesis. L-tyrosine is not an essential amino acid for mammals, since it is synthesized by the hydroxylation of phenylalanine. In mammals, phenylalanine and tyrosine are required for the synthesis of adrenaline, noradrenaline, catecholamine, and dopamine, as well as the thyroid hormones, thyroxine and triiodothyronine, and the pigment melanin. Plants and most microorganisms are able to synthesize phenylalanine via the shikimic acid pathway (Braus, 1991; Hrazdina and Jensen, 1992; Schmid and Amrhein, 1995). The initial stages of phenylalanine biosynthesis (prechorismic acid pathway) are common in plants and microorganisms, as shown in Fig. 1.1. The first step in the biosynthesis of phenylalanine is the condensation of erythrose 4-phosphate and phosphoeno/-pyruvate to form 3-deoxy-D-arao/V70-heptulosonic acid 7-phosphate (DAHP). The cyclization of DAHP to 3-dehydroquinic acid requires cobalt ions and uses bound NAD+ as cofactor. Dehydration of this 3-dehydroquinic acid yields 3-dehydroshikimic acid. 3-dehydroshikimic acid is reduced to shikimic acid in an NADP-l Phosphoeno/ pyruvate + Erythrose 4-phosphate 1 ^ Pi 3-Deoxy-D-araD/>70-heptulosonic acid 7-phosphate (DAHP) 2 k P i 3-Dehydroquinic acid 3 3-Dehydroshikimic acid y- NADPH K\ NADP+ Shikimic acid ^ ATP ADP Shikimic acid 3-phosphate Pyruvate 6 " 5-e/?o/pyruvylshikimic acid 3-phosphate (EPSP) Chorismic acid Fig. 1.1. The pathway from erythrose 4-phosphate to chorismic acid. The enzymes are; 1: 3-deoxy-D-araD/Vjo-heptulosonic acid 7-phosphate synthase, 2: 3-dehydroquinic acid synthase, 3: 3-dehydroquinic acid dehydratase, 4: shikimic acid:NADP oxidoreductase, 5: shikimic acid kinase, 6: 5-e/7o/pyruvylshikimic acid 3-phosphate synthase, and 7: chorismic acid synthase. 2 dependent reaction catalyzed by shikimic acid dehydrogenase. After phosphorylation, shikimic acid 3-phosphate condenses with phosphoeno/-pyruvate. This condensation product, 5-e/7o/pyruvylshikimic acid 3-phosphate, is converted to chorismic acid in a reaction catalyzed by chorismic acid synthase. Beyond chorismic acid, the pathway leading to the synthesis of phenylalanine or tyrosine branched from that which produces tryptophan pathway (Fig. 1.2). In plants, the biosynthesis of phenylalanine proceeds from chorismic acid via prephenic acid and arogenic acid. In fungi, synthesis proceeds from chorismic acid via prephenic acid and either phenylpyruvic acid or arogenic acid. In bacteria, either of these pathways, or sometimes both pathways, can be operative (Bender, 1985; Jensen, 1986). 1.1.2 The metabolic fate of L-phenylalanine 1.1.2.1 In mammals Phenylalanine can be transaminated to phenylpyruvic acid, but this is normally only a minor route of phenylalanine catabolism because the K m of the aminotransferase is relatively high. Phenylpyruvic acid can be further catabolized via reduction to phenyllactic acid, or decarboxylation to phenylacetic acid. 3 Chorismic acid Glutamine r Glutamate + Pyruvate Prephenic acid Anthranilic acid Glutamate a-Ketoglutarate ^ > 3 6 C0 2 + H20 Arogenic acid NADP+ •> NADPH + CO2 Tyrosine Phenylpyruvic acid f Glutamate , ^  C0 2 + H20 J/^  a-Ketoglutarate Phenylalanine Tryptophan COOH Fig. 1.2. The pathway from chorismic acid to aromatic amino acids. The enzymes are; 1: chorismic acid mutase, 2: anthranilic acid synthase, 3: prephenic acid aminotransferase, 4: arogenic acid dehydrogenase, 5: arogenic acid dehydratase, 6: prephenic acid dehydratase, and 7: phenyl pyruvate aminotransferase. Dashed arrows indicate multiple enzyme steps. 4 Under normal conditions, phenylalanine is metabolized by way of hydroxylation to tyrosine. Phenylalanine hydroxylase (a biopterin-dependent mixed function oxidase), dihydrobiopterin reductase, and a stimulator protein, are all involved in this hydroxylation (Fig.1.3). Failure in this hydroxylation process results in phenylketonuria (Ambrus et al., 1978), an inborn metabolic disorder. In affected children, the phenylalanine concentration in blood is very high and tyrosine is low. In almost all cases of phenylketonuria, the defect is in phenylalanine hydroxylase, but a few cases, the defect is an inability to synthesize methyl-biopterin or a lack of dihydropterin reductase (Kaufman et al., 1975, 1978). Under these conditions, a substantial portion of phenylalanine undergoes transamination to phenylpyruvic acid, which is further metabolized to phenylacetic acid and phenyllactic acid. These three phenylalanine metabolites are excreted in the urine. The formation of tyrosine from phenylalanine can lead to catabolism by transamination of tyrosine to yield p-hydroxyphenylpyruvic acid (Fig.1.3). Normally, p-hydroxyphenylpyruvic acid is metabolized by way of homogentisic acid, which is cleaved eventually to fumaric acid and acetoacetic acid as shown in Fig. 1.4. p-Hydroxyphenylpyruvic acid can also be reduced to p-hydroxyphenyllactic acid, however, and this reaction becomes important when the homogentisic acid pathway from p-hydroxyphenylpyruvic acid to maleyl acetoacetic acid is blocked. This block results in alcaptonuria (Bender, 1985), a metabolic disorder that arises from a genetic defect in homogentisic acid oxidase. In 5 o. Phenylalanine ^ 3 Tyrosine 6-Methyltetrahydrobiopterin 4-a-Carbinolamine NADP+ NADPH 1 1 6-Methyl-dihydrobiopterin a-Ketoglutarate Glutamate NADH NAD+ Tyrosine p-Hydroxyphenylpyruvic acid p-Hydroxyphenyllactic acid Phenylalanine 6 J ^ C0 2 Homogentisic acid 0 2 7 ^ Maleyl acetoacetic acid •I Fumaryl acetoacetic acid H20 9/ Fumaric acid Acetoacetic acid Fig. 1.3 The conversion of phenylalanine to tyrosine and other metabolites. The enzymes; 1: dihydropterin reductase, 2: stimulator protein, 3: phenylalanine hydroxylase, 4: tyrosine aminotransferase, 5: p-hydroxyphenylpyruvic acid reductase, 6: p-hydroxyphenylpyruvic acid hydroxylase, 7: homogentisic acid oxidase, 8: maleyl acetoacetic acid isomerase, and 9: fumaryl acetoacetic acid hydrolase. 6 Tyrosine NADH NAD+ 3, 4-Dihydroxyphenylalanine (DOPA) C0 2 Dopamine f 02 Melanin Homovanillic acid Noradrenaline ^ . S-Adenosyl-homocysteine 4 1 , r ^ S-Adenosyl-homomethionine Adrenaline Fig. 1.4. The conversion of tyrosine to catecholamines. The enzymes; 1: tyrosine hydroxylase, 2: DOPA decarboxylase, 3: dopamine p-hydroxylase, and 4: phenylethanolamine-N-methyltransferase. Dashed arrows indicate multiple enzyme steps. 7 alcaptonuria patients, high amounts of p-hydroxyphenyllactic acid are formed and excreted in the urine. The other important metabolic conversion of tyrosine is hydroxylation to 3,4-dihydroxyphenylalanine (DOPA), which is further metabolized to many physiologically important compounds. As shown in Fig. 1.4., DOPA serves as the precursor for the neurotransmitter, dopamine, and for the noradrenaline and adrenaline hormones (Martin, 1972). It also serves as the precursor for melanin formation, a process catalyzed by tyrosinase, a copper-dependent oxygenase, rather than by tyrosine hydroxylase (Giuseppe, 1992). Another important product of mammalian tyrosine metabolism is the thyroid hormone thyroxine and its metabolite triiodothyronine, which stimulate energy metabolism through increasing the rate of electron transport (Metzler, 1977). 1.1.2.2 In plants L-Phenylalanine, derived from the shikimic acid pathway, is used directly for protein synthesis in plants or metabolized through the phenylpropanoid pathway. This phenylpropanoid metabolism leads to the biosynthesis of a wide array of phenylpropanoid secondary products. The first few steps in this metabolic sequence are known as the "general phenylpropanoid pathway" (Fig. 1.5). These steps involve the actions of phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (C4H), and 8 Carbohydrates Primary metabolism Phenylpropanoid metabolism Shikimic acid Chorismic acid Phenylalanine PAL Cinnamic acid C4H 4-Coumaric acid 4CL • 4-Coumaryl-CoA Anthocyanins •* Coumarins -* Flavonoids Lignin -> Stilbenes Suberin \ Tannins Fig. 1.5. Schematic representation of general phenylpropanoid metabolism. Dashed arrows indicate multiple enzyme steps. PAL: phenylalanine ammonia-lyase, C4H: cinnamic acid 4-hydroxylase, and 4CL: 4-coumaric acid: Coenzyme A ligase. 9 4-coumaric acid:CoA ligase (4CL). Following these core reactions, diverse phenylpropanoid products are derived through individual branch pathways (Hahlbrock and Scheel, 1989). These products include anthocyanins, coumarins, furanocoumarins, flavonoids, isoflavonoids, lignin, salicylic acid, stilbenes, suberin, soluble esters, tannins and other phenolics. Many of these phenylpropanoid compounds are known to have functions in allelopathy, color pigmentation, UV-protection, insect repellents, antimicrobial phytoalexins, and signaling in plant-microbe interactions. They also function as cell-wall components, waterproofing agents, and a source of structural rigidity. Some alkaloids also arise from phenylalanine. Examples are colchicine, produced by the Liliaciae family, and ephedrine in Ephedra spp.. In barley, phenylalanine is converted to tyrosine and thence, via tyramine and N-methyltyramine into hordenine (Mann, 1987). Phenylalanine- or tyrosine-derived alkaloids may be grouped as four main structural types. These are simple monocyclic compounds (e.g. mescaline from 'peyote' cactus), isoquinolines (e.g. pellotine from 'peyote' cactus), benzylisoquinolines (e.g. morphine from opium poppy), and Amaryllidaceae alkaloids (e.g. lycorine from the daffodil family) (Mann, 1987). 1.1.2.3 In microorganisms While the metabolism of phenylalanine in vascular plants and animals has been well documented, much less is known about the microbial degradation of phenylalanine. Some of the known pathways of animal and plant metabolism of phenylalanine are also 10 used in microorganisms. In some microorganisms, phenylalanine has often been found to be converted to homogentisic acid through the intermediary formation of phenylpyruvic acid and p-hydroxyphenylpyruvic acid by transamination and hydroxylation, as in the case of animals (Meister, 1965; Wat and Towers, 1979). The discovery of PAL enzyme in fungi (Power et al., 1965) and the detection of 1 4 C0 2 production from 14C-ring-labeled phenylalanine, cinnamic acid, and benzoic acid (Moore and Towers, 1967), demonstrated that certain fungi and Streptomyces bacteria can degrade phenylalanine by a pathway involving an initial deamination to cinnamic acid, as happens in plants. In Streptomyces verticillatus, frans-cinnamamide is formed from trans-cinnamic acid derived from phenylalanine (Bezanson et al. 1970). A metabolic pathway for the metabolism of phenylalanine via cinnamic, benzoic, p-hydroxybenzoic, and protocatechuic acids has been reported in several basidiomycete fungi, including Rhodotorula (Uchiyama et al., 1969), Ustilago hordei (Moore et al., 1967), Schizophyllum commune (Moore and Towers, 1967), and Sporobolomyces roseus (Moore et al., 1968). Schizophyllum commune can also metabolize phenylalanine through phenylpyruvic acid, phenylacetic acid and o-hydroxyphenylacetic acid (Moore and Towers, 1967). Interestingly, it has been reported that another basidiomycete, Lentinus lepideus forms phenylpropanoid compounds (e.g. p-coumaric acid, caffeic acid, isoferulic acid, phloretic acid, and p-methoxycinnamic acid) via cinnamic acid derived from phenylalanine (Towers, 1969). In this fungus, a number of these compounds accumulate in the medium as methyl esters, but the physiological significance of these compounds is not known. The conversion of phenylalanine to benzoic acid derivatives through cinnamic acid has also been reported in Deuteromycete fungi such as Altemaha (Nambudiri et al., 1970), 11 Rhizoctonia solani (Kalghatgi et al., 1974), and Penicillium brevicompactum (Campbell et al., 1987). The fungus Gliocladium produces gliotoxin, an antibiotic and antiviral cyclic peptide, derived in part from phenylalanine and modified by the addition of sulfur across the peptide ring (Griffins, 1994). The conversion of phenylalanine to fungal melanin has received little attention, but was suggested to occur in Alternaria (Pridham and Woodhead, 1977). 1.2 PHENYLALANINE AMMONIA-LYASE (PAL) 1.2.1 Introduction PAL (E.C. 4.3.1.5) catalyzes the nonoxidative deamination of L-phenylalanine to form frans-cinnamic acid and a free ammonium ion (Fig. 1.6) (Koukol and Conn, 1961). As described above, the conversion of the amino acid phenylalanine to frans-cinnamic acid is the entrypoint step for the channeling of carbon from primary metabolism into phenylpropanoid secondary metabolism in plants. PAL has been extensively studied because of its role in plant development and its response to a wide variety of environmental stimuli. The importance of this enzyme in plant metabolism is demonstrated by the huge diversity, and large quantities of phenylpropanoid products found in plant materials (Jones, 1984). In fungi, there is no direct evidence for the significance of this enzyme except as a catabolic function (Marusich et al., 1981). 12 L-histidine trans - urocanic acid Fig. 1.6. Deamination of aromatic amino acids by ammonia-lyases. PAL: L-phenylalanine ammonia-lyase, TAL: L-tyrosine ammonia-lyase, and HAL: L-histidine ammonia-lyase. 13 The presence of PAL has been reported in plants (Koukol and Conn, 1961; Camm and Towers, 1973; Jorrin et al., 1988) including certain algae, e.g. Dunaliella marina (Czichi and Kindl, 1975), fungi (Bandoni et al., 1968; Moore era/., 1968; Hodgins, 1971; Sikora and Marzluf, 1982), and a prokaryotic organism, Streptomyces verticillatus (Ernes and Vining, 1970). In plants, PAL activity has been detected in many species, representing monocots, dicots, gymnosperms, ferns, and lycopods (Young et al., 1966). In fungi, PAL activity has been detected only in a few basidiomycetes and deuteromycetes, and in one ascomycete, Nectria cinnabarina (Bandoni et al., 1968; Vance et al., 1975). There have been no reports of PAL in animals. 1.2.2. Commercial and medical potential of PAL The therapeutic potential of using PAL enzyme against neoplasms has been suggested because of its selectivity for phenylalanine (Stith et al., 1973). PAL substantially inhibited neoplastic cell growth in vitro (Abell et al., 1972), and produced cures in some mice that were inoculated with a lymphoblastic leukemia (Abell et al., 1973). However, PAL is of special interest to clinicians primarily due to its potential as a treatment for the inherited metabolic disorder, phenylketonurea. A treatment involving the oral ingestion of PAL (Hoskins et al., 1980) were proposed to patients to consume a normal diet, but this has not been brought to clinical trials yet. With the increasing consumption of the aspartic acid-phenylalanine dipeptide artificial sweetener, Aspartame, the commercial demand for L-phenylalanine has led to mass 14 production of this amino acid (Klausner, 1985). Since the reaction is reversible, PAL can be used in a large-scale bio-conversion to produce L-phenylalanine from frans-cinnamic and ammonium salts acid (Hamilton era/., 1985). 1.2.3 Active site and enzyme mechanism PAL is one of the few amino acid-transforming enzymes not containing the cofactor pyridoxal 5'-phosphate. Instead, PAL contains the unusual prosthetic group dehydroalanine (Hodgins, 1971). The role of this post-translationally modified amino acid in catalysis is assumed to be the activation of the amino group of phenylalanine to form a better leaving group than NH3+ (Hanson and Havir, 1972). Modification of an electrophilic center at the active site of PAL by electrophilic reagents such as borohydride, cyanide, bifulfite or nitromethane, results in the complete inactivation of the enzyme. The identity of [3H]-alanine and [14C]-aspartic acid released following acid hydrolysis of PAL enzyme inactivated with radiolabeled reagents, NaB3H4 and 14CN", provide evidence for the presence of dehydroalanine in the active site (Hanson and Havir, 1985; Hodgins, 1971). Studies on the ability of substrates and substrate analogs of PAL to prevent inactivation by these reagents provide further evidence to support the idea that the active site contains dehydroalanine (Hanson, 1970; Hodgins, 1971). Recently, an alternative model for the role of dehydroalanin in PAL catalysis has been proposed (Schuster and Retey, 1995) The mechanism of formation of dehydroalanine has not been determined yet. In cases of other proteins which contain dehydroalanine e.g. subtilin (Banerjee and Hansen, 1988), 15 thyroglobulin (Ohmiya et al., 1990), and pyruvoyl enzymes (Recsei and Snell, 1984), a serine residue is considered to be the precursor of dehydroalanine. PAL amino acid sequences contain a serine residue which is completely conserved among different species (Taylor et al., 1990), and is presumed to be associated with the active site of the enzyme. Recently, the precursor of the dehydroalanine residue has been identified as serine in parsley (Schuster and Retey, 1994) and poplar (McKegney et al., 1996) PAL. In fungal PAL, it is likely that a similar process would account for the formation of the active site dehydroalanine from serine, but a role for serine as the precursor of dehydroalanine has not been directly demonstrated yet. Expression of PAL in Escherichia coli (Schulz et al., 1989; Orum and Rasmussen, 1992) produced active PAL enzymes in cells in which PAL is not normally produced. The expressed PAL proteins showed similar enzyme properties compared to endogenous PAL from other sources. This suggests that the formation of dehydroalanine may be an autocatalytic process, although it cannot be ruled out that a widespread modifying enzyme is involved in the dehydroalanine formation. 1.2.4 Structural properties 1.2.4.1 PAL proteins PAL has been isolated and characterized from a number of plant species, some fungi and one bacterial source. Source tissues used for PAL isolation are diverse. They include seedlings (Nari et al., 1972), shoots (Koukol and Conn, 1961), leaf-sheath (Havir and Hanson, 1973), cell culture (Jorrin et al., 1988; Bernards and Ellis, 1991; Campbell and Ellis, 1992), fruit (Given et al., 1988), and mycelium (Pridham and Woodhead, 1974; 16 Kalghatgi and Subba Rao, 1975). Most known PAL sources for enzyme isolation and its properties are well tabulated and documented in reviews by Camm and Towers (1973), Hanson and Havir (1981), and Schomburg and Salzmann (1990). Difficulties in purification are often encountered, partly resulting from the low abundance of PAL in cells and changes in size and properties that occur during purification. Although an apparently homogeneous protein preparation can often be obtained in non-denaturing conditions, additional polypeptide bands are usually detected in analytical PAGE gel under denaturing conditions. This can create confusion in the estimation of PAL subunit sizes. Most PALs reported range in size from 300 to 340 kDa in native molecular mass. Some examples of exceptions are reported masses of 152 kDa in Ocimum basilicum (Hao er a/., 1996), 226 kDa in a bacterium, Streptomyces (Ernes and Vining, 1970), 250 kDa in Helianthus annuus (Jorrin et al., 1988), 266 kDa in Fragaria ananassa (Given et al., 1988), and 560 kDa in Altemaria (Pridham and Woodhead, 1974). PAL is normally a homo-tetrameric protein consisting of 4 identical subunits. Hetero-tetrameric PAL as a complex of two hetero-dimers has been reported from Helianthus annuus (2 x 58 kDa and 2 x 68 kDa, Given et al., 1988) and Rhizoctonia solani (2 x 70 kDa and 2 x 90 kDa, Kalghatgi and Subba, 1975). Neumann and Schwemmle (1993) reported that Oenothera seedlings have two PAL isoenzymes with four identical subunits each of 75.5 kDa and 79.2 kDa. Rhodosporidium toruloides PAL has been reported to be a dimer being composed of two identical subunits with a mass of 80 kDa (Adachi et al., 1990). 17 Iso-electric points (pi) for PAL are usually in the acid range from 2.5 (Neumann and Schwemmle, 1993) to 5.75 (Campbell and Ellis, 1992). Isoforms with different pis have been reported from some sources; three isoforms ranged between pi 4.8 and 5.4 in Leptosphaeria maculans (Dahiya, 1993), several isoforms between pi 5.1 and 6.1 in alfalfa (Jorrin and Dixon, 1990), and two isoforms between pi 4.8 and 5.4 in bean (Bolwell and Rodger, 1991). Interestingly, expression of a single cDNA of poplar PAL in a baculovirus expression system produced two isoforms with different pis (McKegney et al., 1996). Most PALs are considered to be hydrophobic proteins. This property has led to the use of hydrophobic affinity column chromatography for the purification of PAL from cotton (Dubery and Smit, 1994) and Rhodotorula glutinis (D'Cunha et al., 1996). Alfalfa PAL has been reported to be highly hydrophobic (Jorrin and Dixon, 1990) and consistent with this, the hydropathy profile of the protein sequence deduced from the cDNA sequence also predicted that alfalfa PAL would be hydrophobic (Gowri et al., 1991). The association of carbohydrate with PAL has been reported for the maize and potato enzymes (Havir, 1979; Shaw et al., 1990). Through the analyses of PAL gene sequences, the presence of potential glycosylation sites has been reported from bean (Cramer et al., 1989) and parsley (Lois et al., 1989), but the importance of glycosylation in PAL function has not been explored extensively. The production of active PAL in E. coli cells transformed with PAL genes from the yeast Rhodosporidium (Orum and Rasmussen, 1992) and from parsley (Shulz et al., 1989; Appert et al., 1994) suggests that PAL catalysis is not likely to be influenced by glycosylation. It has not been excluded 18 that glycosylation is involved in enzyme stability and in localization of the enzyme within cells (Havir, 1979; Shaw era/., 1990). 1.2.4.2 PAL-encoding genes Following the isolation of PAL cDNA from bean (Cramer et al., 1989), parsley (Lois et al., 1989) and sweet potato (Tanaka et al., 1989), PAL genes have been isolated from many sources. Currently, information on the partial or full sequences of the genes and cDNAs encoding PAL is available from several nucleic acid databases. In most plants, PAL is encoded by a small gene family of 3-5 genes. Exceptions to this are the potato PAL gene family which is made up of 40-50 genes (Joos and Hahlbrock, 1992), and the loblolly pine PAL which has been reported to be encoded by a single gene (Whetten and Sederoff, 1992). In yeasts, PAL is encoded by single gene (Anson et al., 1987). In general, the sizes of PAL genes have been reported in the range from 2.1 kb to 2.4 kb. The presence of introns has been reported both in plant and yeast PAL genes. Plant PAL genes generally contain only one intron, while yeast PAL genes have five (Anson et al., 1987) or six introns (Valslet et al., 1988). There are two exceptions; two introns have been found in the Arabidopsis PAL gene (Wanner et al., 1995), while no introns occur in jack pine and loblolly pine PAL genes (Campbell, 1991; S. Butland, personal communication ; Sederoff era/., 1994). 1.2.4.3 PAL promoter 19 The organization of PAL promoters has been studied by analyses of the 5' flanking regions of plant PAL genes using DNA footprinting, S1 mapping, and primer extension methods. The presence of a TATA box in PAL gene promoters has been reported from bean (Cramer et al., 1989), parsley (Lois et al., 1989), and rice (Minami et al., 1989). Bean and rice PAL promoters contain a CAAT box, and a GC-rich segment is present in the rice PAL promoter. As in the cases of some other phenylpropanoid enzyme-encoding genes e.g. 4CL and CHS (Dron et al., 1988; Zhang and Mehdy, 1994), there are H [CCTACC(N)7CTJ and AC Boxes upstream from the PAL transcription start site (Hatton et al, 1995; Loake et al., 1992; Lois et al., 1989). However, none of these sequences were found in the upstream region of a yeast PAL gene (Anson et al. ,1987). 1.2.5 Functional properties PAL enzymes from many sources, especially from monocots and certain fungi, have activity towards L-tyrosine and can thus produce frans-p-coumaric acid. This has been described as tyrosine ammonia-lyase (TAL, Fig. 1.6) activity (Neish, 1960; Young et al., 1966, Camm and Towers, 1973). In most PAL preparations, TAL activity is very low. The PAL/TAL ratio in PAL preparations varied from 1.35 to 5 in Sporobolomyces pararoseus (Parkhurst and Hodgins, 1971), from 4 to 20 in wheat (Young et al., 1966), and from 0.6 to 1.3 in bean (Scott et al., 1992). An even larger range in PAL/TAL ratios from several different plant species was reported by Jangaard (1974). No TAL enzyme without PAL activity has been purified. Recently, it has been demonstrated in E. co//-expressed maize PAL that PAL and TAL activities reside in the same polypeptide (Rosier et al., 1997). 20 PAL preparations from a number of sources are reported to have only one Michaelis constant ( K m ) , but the kinetic properties of other preparations suggested that the enzyme is negatively cooperative with respect to substrate binding (Nari et al., 1974). Two different K m values for PAL have therefore been reported from many sources (Hanson and Havir, 1981). However, when individual isoforms were highly purified, kinetic analysis of each isoform revealed normal Michaelis-Menten saturation kinetics (Bolwell et al., 1985; Jorrin and Dixon, 1990). The K m values for L-phenylalanine have been reported to range from 0.011 mM (Nagai et al., 1988) to 1.7 mM (Koukol and Conn, 1961). Most PAL shows no metal ion requirement although slight stimulation of PAL activity by metal ions such as Mg2+ and Ba2+ has been reported (Jorrin et al., 1988). Inhibition of PAL activity can be induced by a wide range of compounds including carbonyl, sulfhydryl and thiol reagents, phenolic acids and heavy metal ions (Schomburg and Salzmann, 1990). Most PALs tested are sensitive to the synthetic PAL inhibitors such as (S)-2-aminooxy-3-phenylpropanoic acid (AOPP), (R)-(1-amino-2-phenylethyl)phosponic (APEP) acid, and 2-aminoindan-2-phosphonic acid (AIP) and thus these inhibitors have often been used to block the biosynthesis of phenylpropanoid compounds in plant cells and tissues (Hanson and Havir, 1981, Zon and Amrhein, 1992). The pH optimum for PAL is generally in the range from 8.2 to 9.0 (Pridham and Woodhead, 1974; Hanson and Havir, 1981). The temperature optimum for PAL has been reported to be 35°C in tobacco (Nagai et al., 1988), 55°C in sunflower (Jorrin er al., 1988), and 44-46°C in Rhizoctonia (Kalghatgi and Subba Rao, 1975). Plant PAL enzymes are generally sensitive to repeated freezing and thawing and lose activity as the temperature approaches 60°C. In contrast, fungal PAL is more thermally stable too 21 (Kalghatgi and Subba Rao, 1975). Rhodotorula PAL is apparently stable for at least six months when it is kept at -60°C (Fritz et al., 1976). 1.2.6 Regulation PAL enzyme activity is generally regulated at two levels - PAL synthesis, and inactivation or degradation. PAL synthesis is determined through the processes of transcription, transcript processing, and translation. PAL inactivation or degradation is mediated by several factors such as stability of enzyme, proteolytic enzyme, inhibitors, and modification of the enzyme. The rate of each process and the degree of mediation by each factor described above can affect the levels of PAL activity. These modes of PAL activity regulation are reviewed in the following sections (1.2.6.1-3). 1.2.6.1 Factors influencing PAL activity levels PAL activity levels in plants are influenced by several environmental factors including light, pathogen attack, wounding, temperature, oxygen (Hanson and Havir, 1981; Jones, 1984). The levels also depend on the genotype, plasmotype, age, organ and tissues of the plant (Camm and Towers, 1973). In microorganisms, by contrast, there is almost no information on environmental factors involved in modulating PAL activity levels. Light generally has a stimulatory effect on PAL activity levels although exceptions have been reported. An increased level of extractable PAL activity in response to wounding has been displayed in many plant species including bean axes, pea seedlings, potato 22 and sweet potato tubers, and sunflower leaves (Camm and Towers, 1973; Hanson and Havir, 1981; Shaw et al., 1990). In diseased plants, increased synthesis of phenolics, lignin and isoflavonoid phytoalexins is concomitant with the increased PAL activity (Friend, 1981; Vance era/., 1980; Grisebach and Ebel, 1978). Hormonal effects on PAL activity also have been demonstrated, and these may link responses to pathogen infection with those associated with plant development. Endogenously produced ethylene appears to trigger increased PAL production in bean and tobacco infected with viruses, and in parsley, rice, sweet potato, and soybean infected with fungal pathogens (reviewed by Camm and Towers, 1973; Chappell et al., 1984; Haga et al., 1988). Inhibition of PAL induction by indole acetic acid (IAA) and abscisic acid has been reported in tobacco tissue cultures and soybean seedlings infected with a fungal pathogen, respectively (Innerarity et al., 1972; Ward era/., 1989). 1.2.6.2. Mechanisms for the regulation of PAL activity There are many examples of PAL activity regulation at the protein level. Up-regulation could involve enhancement of de novo PAL synthesis and substrate pools, conversion of PAL from inactive to active forms, reduction of active PAL degradation, and/or an increase in enzyme turn-over. Among these mechanisms, the major mechanism for the increase of PAL activity levels appears to be increased de novo synthesis of PAL proteins. De novo PAL synthesis in response to environmental stimuli has been demonstrated by radio-labeling of newly synthesized PAL or by use of a protein synthesis inhibitor, cycloheximide (Lawton et al., 1983; Hahlbrock and Ragg, 1975). 23 Down-regulation of PAL activity levels would be possible through reversal of the processes responsible for up-regulation. Namely, reduction of new PAL synthesis and depletion of substrate pools, and an increase in the inactivation and/or degradation of PAL enzyme. The direct inactivation of PAL activity by proteinaceous factors has been described in extracts from apple skin (Tan, 1980), sunflower leaves (Gupta and Creasy, 1984), and sweet potato (Tanaka et al., 1977). Bolwell (1992) has indicated that cAMP-dependent phosphorylation could be involved in suppressing the induction of PAL activity. In Phaseolus, phosphorylation of PAL subunits was associated with a smaller subunit size (70 kDa), but with the normal size of subunit (77 kDa), suggesting that PAL degradation and inactivation may be triggered by phosphorylation of the enzyme. Bolwell and his colleagues (1986,1988) have reported that in bean suspension cultures, cinnamic acid can decrease the levels of PAL activity by increasing the degradation of PAL. In tomato, in contrast, it has been proposed that production of truncated gene products, which would presumably yield disfunctional PAL proteins, might lead to changes in PAL activity (Lee et al., 1992). 1.2.6.3. Regulation of PAL gene expression PAL activity is commonly regulated at the genetic level in response to various stimuli. PAL gene expression has been observed to be activated in plants infected with tobacco mosaic virus (Pellegrini et al., 1994), bacteria (Huang and McBeath, 1994), and fungi (Bell et al., 1986; Bernards and Ellis, 1991; Lamb et al., 1989; Joos and Hahlbrock, 1992). Many of the responses to pathogen attacks can be mimicked by elicitors derived 24 from fungal cell walls or culture filtrates (Edwards et al., 1985; Rohwer et al., 1987; Dixon and Lamb, 1990). It has been shown that elicitor treatments can also induce PAL gene expression in bean (Shufflebottom et al., 1993), parsley (Dangl et al., 1987; Lois et al., 1989), and in suspension cultured cells of alfalfa (Orr et al., 1993; Farrendorf et al., 1996), rice (Zhu et al., 1995) and poplar (Moniz de Sa et al., 1992). Hormonal effects on PAL gene expression in association with pathogen infection or elicitor treatments have been reported. ABA repressed the accumulation PAL mRNA in fungal pathogen-infected soybean (Ward et al., 1989), while in bean suspension cultures treated with elicitor, auxin and ethylene increased the levels of PAL mRNA (Hughes and Dickerson, 1989). Ethylene treatment increased PAL mRNA accumulation in carrot suspension cultures (Eckeref al., 1987). Parsley is one of the best studied plants with respect to the relationship between PAL gene expression and illumination. A rapid transient increase of PAL transcripts has been observed in parsley cell suspension cultures (Betz et al., 1978; Shroder et al., 1979) and protoplasts (Ohl et al., 1990) following UV irradiation. Accumulation of PAL mRNA in tissues exposed to light has been demonstrated in dark-grown parsley seedlings which were then transferred to the light (Wu and Hahlbrock, 1992). Accumulation of PAL mRNA has also been observed in Arabidopsis seedlings illuminated by UVB or blue light (Kubasek etal., 1992) and hybrid poplar leaves irradiated by UV (Osakabe et al., 1995). Liang and his colleagues (1989) have shown that in etiolated transgenic tobacco plants, white light increases GUS activity which is expressed from a PAL promoter-GUS fusion transgene. 25 The response of PAL genes to wounding and heavy metals has also been studied. Increased PAL gene transcription in response to wounding has been detected in Arabidopsis (Ohl et al., 1990), melon (Diallinas and Kanellis, 1994), parsley (Lois and Hahlbrock, 1992), and sweet potato (Tanaka et al., 1989). PAL genes in Arabidopsis and pea respond to HgCI2 and CuCI2 exposure, respectively, with enhanced mRNA production (Ohl et al., 1990; Preisig et a/.,1991). PAL gene expression is also regulated developmentally and in a tissue-specific fashions. During stem growth in Populus kitakamiensis, PAL genes PAL g1 and PAL g2a were expressed in a tissue-specific manner. The highest levels of PAL g1 were detected in young tissue near the shoot bud, but PAL g2a mRNA was mainly accumulated in mature stems (Osakabe et al., 1995). PAL transcript changes in association with development of vascular tissue and other tissue-specific patterns of expression have been observed in other plant systems including alfalfa, Arabidopsis (Wanner et al., 1995), bean (Leyva et al., 1992), parsley (Wu and Hahlbrock, 1992), and pea (Yamada et al., 1994). In melon fruit, PAL is transcriptionally induced in response to fruit ripening (Diallinas and Kanellis, 1994). The analyses of tissue-specific expression of the bean PAL2 and PAL3 promoters in transgenic tobacco have revealed that the AC elements play a key role in regulation of tissue-specific expression of the bean PAL gene family (Hatton et al., 1995,1996). 26 1.2.6.4 Co-ordinate regulation It has been shown that, like other phenylpropanoid enzymes and their genes, expression of PAL enzyme is coordinately regulated with other enzymes of the phenylpropanoid pathway (reviewed by Logemann et al., 1995). Cramer et al. (1985) have reported that PAL, chalcone synthase (CHS), and chalcone isomerase (CHI) are coordinately regulated in elicitor-treated bean cultures. Co-ordinate regulation of PAL and CHI has also been observed in illuminated mustard seedlings (Beggs et al., 1987). In parsley cell suspension cultures, UV irradiation and elicitor treatment produced a coordinate increase in PAL and 4CL transcript levels (Chappel and Hahlbrock, 1984; Schmelzer et al., 1985). A similar coordinate expression of PAL and 4CL mRNAs in response to elicitor has been observed in alfalfa (Dalkin et al., 1990) and potato (Fritzemeier et al., 1987). The expression of PAL with 4CL and with other pathogenesis-related (PR) and elicitor-related (EL) genes is regulated coordinately in parsley leaves challenged with a fungal pathogen (Schmelzer et al., 1989). The observation that the AC elements found in both the PAL1 and CHS promoters are involved in activation of transcription in parsley (Lois et al., 1989; Schulze-Lefert et al., 1989), provides a possible mechanism by which phenylpropanoid metabolism could be coordinately regulated (Cramer era/., 1989; Logemann era/., 1995). 27 1.3 USTILAGO MAYDIS 1.3.1 Corn smut disease Smut fungi are basidiomycetes of the order Ustilaginales. These fungal pathogens cause severe economic losses of cereal grains (such as barley, corn, millet, oats, sorghum, and wheat), other crops (such as onions, spinach), and some ornamentals (such as carnation). The most common smuts belonging to the genus Ustilago are smuts of barley (U. hordei, U. nuda, U. nigra), corn (U. maydis), oats (U. avenae), sugarcane (U. scitaminea), and wheat (U. tritici). The infection of corn (Zea mays) by U. maydis is characterized by distinct galls (plant tumors) on stems, leaves, ears, and tassels (Christensen, 1963). The disease symptoms also include stunting, and chlorosis and anthocyanescence on stems and leaves, especially in the areas developing tumors . The fungus reproduces primarily in embryonic and protein-rich tissues of its host, and stimulates its host cells to divide and enlarge. In the end, this proliferation produces tumors containing dark, sooty, masses of black teliospores, from which the name of the disease, corn smut, is derived. U. maydis is distributed world-wide, but is particularly prevalent in warm and moderately dry areas, where it causes serious damage to susceptible varieties (Agrios, 1988). Losses from this disease vary from one area to another, but can reach 10% or more. Control of the disease is mainly reliant on use of resistant corn varieties, and to a lesser extent on sanitation and crop rotation measures. In addition, the maintenance of well-balanced soil fertility can reduce disease incidence (Ullstrup,1961). 28 1.3.2 The life cycle and mating of U. maydis Several phases of the life cycle of U. maydis are outlined in Fig. 1.7. The fungus produces two types of cells in its life cycle. The unicellular haploid phase is saprophytic (non-pathogenic), grows by budding, and forms compact colonies with yeast-like morphology. This cell type can be easily cultured in vitro and produces a colony of sporidia by budding. Another type of cell is the dikaryon which is pathogenic, parasitic and dependent on growth in host tissues by infection. The dikaryon arises from mating between two compatible haploid sporidia. Through karyogamy (nuclear fusion) within the dikaryotic hyphae inside the host plant, diploid cells are produced which develop into black teliospores within galls. The teliospores are released from ruptured plant galls and dispersed by wind. After germination of teliospores, meiosis occurs and haploid sporidia are produced. Compatible mating of these sporidia leads to a new life cycle of U. maydis (Banuett, 1992). The intertwined association of life cycle and pathogenicity has provided opportunities to explore the processes that govern mating, filamentous growth, and pathogenicity. It has been found that to undergo the pathogenic process involving infection and development of disease symptoms, fusion of two compatible yeast-like haploid ceils is essential. Compatibility is determined by the alleles present at two different mating-type loci, the a locus with two alleles (a 1 and a2), and the b locus with multiple alleles (Rowell and 29 Growth of dikaryotic hyphae and gall formation Infection of host plants Karyogamy and formation of teliospores Dikaryotic infectious hyphae Fusion of sporidia Sporidia bud in yeast-like manner Haploid sporidia Diploid teliospores spread by wind Germination, meiosis Fig. 1.7. Schematic representation of the life cycle of Ustilago maydis. 30 Devay, 1954; Puhalla, 1968, 1969; Day et al., 1971). Considerable progress has been made in clarifying the mode of action of these different mating loci. The a mating-type locus, encoding a pheromone (at) and a pheromone receptor (a2), has been determined to be involved in the control of haploid cell fusion (Bolker et al., 1992). The b mating-type locus apparently has at least 25 alleles, and controls pathogenicity and dimorphism (Rowell and Devay, 1954; Day et al., 1971). Two genes are found at the b locus, bE and bW, each encoding a protein containing a homeodomain motif (Kronstad and Leong, 1990; Schulz et al., 1990). It is thought that b gene products have a DNA-binding function, leading to the idea that interactions between the products of the bE and bW genes from alleles of different b specificities are required for pathogenic development in this smut organism (Gillissen et al., 1992). 1.3.3 U. maydis as a model fungus The phytopathogenic fungus U. maydis has been used to study many aspects of eukaryotic genetics, including recombination mechanisms (Holliday, 1974), nitrogen metabolism (Lewis and Fincham, 1970), and developmental regulation (Froeliger and Kronstad, 1990). Classical genetic approaches have been productive with this fungus for several reasons. Both yeast-like haploid and diploid cell cultures can be maintained on nutrient media; teliospores germinate in vitro, making analysis of meiotic products reliable, and diploid strains can be constructed for the study of genetic complementation and dominance (Puhalla, 1968, 1969; Holliday, 1974). Furthermore, molecular genetic approaches are also possible in U. maydis, with the development of such molecular tools 31 as efficient transformation (Wang et al., 1988), autonomously replicating vectors (Tsukuda et al., 1988), genes for selection markers (Wang et al., 1988; Banks and Taylor, 1988; Gold et al., 1994b), and gene disruption (Kronstad et al., 1989). These techniques allow genes of interest to be cloned, manipulated, silenced and/or reintroduced at a desired location in the genome. These resources have greatly facilitated analysis of the structure, expression, and function of U. maydis genes (Froeliger and Kronstad, 1990). 32 1.4 RATIONALE AND OBJECTIVES While a huge amount of information has been accumulated on the structure, expression and function of PAL in plants, the biological role of PAL in fungi has not been established, and, in general, information on fungal PAL is very limited. Most commonly, a catabolic function for fungal PAL has been suggested, in which the enzyme is used to obtain carbon and nitrogen from external supplies of amino acids. However, fungi can also obtain carbon and nitrogen from L-phenylalanine through phenylalanine aminotransferase or amino acid oxidase, which raises the question: what selective advantage does PAL offer that has led to its retention in this group organisms? It appears that the ability to synthesize cinnamic acid is important in the life cycle of fungi. The rationale for this work was to develop knowledge and tools that would enable me to rationalize the existence of PAL in the fungus U. maydis. As mentioned in the previous section, U. maydis is both an agriculturally important fungus and a very versatile model species in which to study the structure, expression, and function of eukaryotic genes. Once it was established that U. maydis also produces PAL, I undertook detailed structural, functional and metabolic studies of this PAL. This work enabled me to clone the U. maydis PAL gene, which make it possible to consider gene replacement. This should reveal whether PAL is essential in Ustilago physiology and/or especially reveal links, if any, between PAL activity and pathogenesis. The following chapters describe the work that was carried out to these ends. 33 Chapter two describes the purification and characterization of the U. maydis PAL enzyme. U. maydis PAL protein data and anti-PAL antibodies were important tools that would provide a unique opportunity to pursue molecular genetic studies of PAL in a fungal species largely unrelated to the red yeasts. Chapter three describes the isolation and sequencing analysis of a U. maydis genomic clone which contains a gene encoding PAL. A comparative analysis of its predicted protein sequence with some of the published PAL sequences, and with sequences for a related enzyme, histidine ammonia-lyase (HAL), provided direct evidence for the extent of evolutionary divergence in this family. Immunological evidence regarding the relatedness of the two ammonia-lyases is also presented. Chapter four describes the in vivo regulation of U. maydis PAL by L-tryptophan, and provides an initial look at L-phenylalanine metabolism in this fungus. A biosynthetic pathway for Ustilago melanin formation is proposed and potential biological roles for Ustilago PAL are discussed in this context. 34 CHAPTER TWO Purification and Characterization of Phenylalanine Ammonia-lyase from Ustilago maydis 2.1. INTRODUCTION Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) catalyzes the nonoxidative deamination of L-phenylalanine to form frans-cinnamic acid and a free ammonium ion. The enzyme occurs in plants (Koukol and Conn, 1961; Camm and Towers, 1973; Jorrin et al., 1988), fungi (Bandoni et al., 1968; Moore et al., 1968; Hodgins, 1971; Sikora and Marzluf, 1982), and Actinomycetes (Ernes and Vining, 1970), but not in animals. PAL has been used in experimental treatments and diagnosis of phenylketonurea, a human syndrome characterized by high levels of circulating phenylalanine (Ambrus et al., 1978), and the enzyme has industrial application in the production of L-phenylalanine from trans-cinnamic acid, i.e. the reverse of the normal in vivo reaction (Yamada et al., 1981). In plants, PAL is the entrypoint enzyme into phenylpropanoid metabolism and it regulates the biosynthesis of a wide range of phenylpropanoid secondary compounds, including lignin, flavonoids, furanocoumarin and isoflavonoid phytoalexins, and wound protectant hydroxycinnamic acid esters (Jones, 1984). Ustilago maydis is the causal agent of corn smut. The fungus reproduces primarily in embryonic and protein-rich tissues of its host (Zea mays), and produces galls on any above-ground part of the host plant (Agrios, 1988). The presence of PAL in the related 35 species, U. hordei, was reported earlier by Subba Rao et al. (1967), but nothing is known about the role of PAL in the Ustilago life cycle or pathogenesis. In a yeast, Rhodosporidium toruloides, phenylalanine can act as the sole source of carbon, nitrogen, and energy (Marusich et al., 1981). Since PAL catalyzes the initial reaction in the catabolism of the amino acid in this organism, the enzyme plays a key role in regulating phenylalanine-dependent metabolism. However, the biological function of PAL in R. toruloides, U. maydis and other fungi during normal (i.e. non-phenylalanine-dependent) growth and development is unclear. It is now possible to conduct gene replacement and gene disruption experiments in U. maydis (Kronstad et al., 1989), and these techniques provide a unique opportunity to address the role of PAL. In a preliminary study, it has been established that, like U. hordei, cultured cells of U. maydis grown in synthetic medium produce substantial amounts of PAL, and the present chapter describes the purification and characterization of the U. maydis enzyme. 2.2 MATERIALS AND METHODS 2.2.1 Materials DIG-Glycoprotein Detection kit and pi markers were purchased from Boehringer Mannheim (Laval, Canada). Molecular mass markers for native PAGE and HiTrap-NHS matrix were purchased from Pharmacia Biotechnology Products (Uppsala, Sweden). DEAE cellulose and Freund's adjuvant were purchased from Sigma (St. Louis, U.S.A.). Bio-Gel A-0.5m agarose beads, Ampholyte, alkaline phosphatase-conjugated goat anti-36 rabbit antibody, SDS-PAGE molecular markers, and BCIP (5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt)/NBT (p-nitro blue tetrazolium chloride) were purchased from Bio-Rad (Mississauga, Canada). Westran PVDF (polyvinylidine difluoride) membrane and Centriprep Concentrator were purchased from Schleicher & Schuell (Keene, U.S.A.) and Amicon (Beverly, U.S.A.), respectively. 2.2.2 Fungal cultures Ustilago maydis strain 518 was maintained on potato dextrose agar medium (PDA) and grown in complete liquid medium (cm) as described by Holliday (1974). For enzyme isolation, cm (50 ml) in 250 ml Erlenmeyer flasks was inoculated with sporidia of the fungus grown on PDA and incubated at 30°C for 24 hr on a gyratory shaker (250 rpm). 2.2.3 Purification of U. maydis phenylalanine ammonia-lyase All procedures were carried out at 4°C, unless otherwise mentioned. The fungal cells were harvested by centrifugation (10000 x g, 10 min), washed twice with distilled water, frozen in liquid nitrogen and stored at -70°C until needed. Cells (30 g) were mixed with 60 g alumina and homogenized for 30 min in a mortar and pestle. The homogenate was extracted with 120 ml 50 mM sodium phosphate buffer (pH 8.0) by stirring for 1 hr, and centrifuged (20000 x g, 30 min). The supernatant (115 ml) was heated at 50°C for 10 min, cooled in ice and centrifuged (20000 x g, 30 min). Protamine sulfate (2%, pH 7.0) was slowly added to the supernatant (final concentration 0.1%) and stirred for 30 min. 37 After centrifugation (20000 x g, 30 min), the pellet was discarded, and the supernatant was fractionated between 30% and 60% saturation with (NH4)2S04. The pellet was dissolved in 10 ml buffer A (1 mM sodium phosphate, pH 7.0) and dialyzed against 3 L buffer A (16 hr with 3 changes). The centrifuged dialyzate was applied to a DEAE cellulose column (2 x 28 cm), washed with buffer A, and eluted with buffer A containing NaCI in a linear 0-0.5 M gradient. The highest PAL activity fractions were pooled and brought to 70% (NH4)2S04 saturation. After centrifugation, the pellet was dissolved in 4 ml buffer A, dialyzed, centrifuged, and concentrated on a Amicon Centriprep concentrator. The concentrate (1 ml) was applied to a Bio-Gel A-0.5m column (1 x 110 cm) and eluted with 100 ml buffer A. The highest PAL activity fractions were combined, concentrated, and dialyzed. A cooled 6% preparative vertical polyacrylamide slab gel was used for the final purification step (see below for electrophoresis). Two 0.5 cm wide vertical strips were excised from the center of each gel. One was silver stained and the other was divided into 0.5 cm wide segments. Each segment was minced separately in 300 ul K-borate buffer (pH 8.8), shaken for 30 min, ground with a mini-pestle, centrifuged, and assayed for PAL activity and protein. 2.2.4 Enzyme assay PAL activity was measured radiometrically as previously described (Bernard and Ellis, 1989) using L-[U-14C]-phenylalanine as substrate. Protein concentration was estimated by the dye-binding method (Bradford, 1976), with bovine serum albumin as standard. All assays were done in triplicate. To test the effect of inhibitors and activators on in vitro activity of PAL, Bio-Gel purified PAL in PAL assay buffer was mixed with compounds to 38 be tested. After a 20 min incubation, the mixture was filtered through a Sephadex G25 column and assayed with the standard reaction mixture. For the detection of tyrosine ammonia-lyase (TAL) activity, crude and Bio-gel purified PAL enzyme preparations were assayed by the spectrophotometric method and by the radiometric method using tritium-labeled L-tyrosine as substrate. 2.2.5 Polyacrylamide gel electrophoresis Vertical slab gels were used for all PAGE analyses. Polyacrylamide gels (5, 6, 7 and 8% (w/v)) were prepared for native PAGE and run with the Laemmli discontinuous buffer system (Laemmli, 1970) in which SDS was omitted from the loading and running buffer. To determine native molecular weight (Mr) of PAL protein, the mobility of PAL at each gel concentration was compared with that of thyroglobulin (660 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and BSA (67 kDa). Subunit Mr was determined by mobility in 7.5% SDS-PAGE gels, compared to that of myosin (205 kDa), p-galactosidase (116 kDa), phosphorylase b (97 kDa), BSA (66 kDa) and ovalbumin (43 kDa). Separated protein bands were detected by Coomassie Brilliant Blue R-250 or silver staining. To detect PAL charge isoforms, the purified PAL was run in a native IEF-PAGE system (Robertson et al., 1987) using pH 3-10 Ampholyte, and silver stained. For isoelectric point (pi) determination, the mobility of the PAL band was compared with that of cytochrome c (horse heart, pi 10.6), myoglobin met (whale sperm, pi 8.3), myoglobin met (horse, pi 7.3), myoglobin met (porcine, pi 6.45), trifluoracetylated myoglobin met (porcine, pi 5.92), azurin (P. aeruginosa, pi 5.65,) and C-phycocyanin (A nidulans, pi 4.75 and 4.85). 39 2.2.6 Antibody production and immuno-affinity chromatography PAL protein-containg bands excised from preparative PAGE gels were minced in 0.5 ml Freund's adjuvant and New Zealand White rabbits were immunized with one subcutaneous and three intramuscular injections of 50 ug PAL at 10 day intervals. Specificity of the antiserum was assayed by double diffusion in agar and the titer was determined by dot immuno-blot assay. The y-globulin fraction was isolated from U. maydis PAL antiserum by DEAE cellulose chromatography (Johnston and Thorpe, 1982). After incubation of a mixture of purified U. maydis PAL and the y-globulin fraction at 25°C for 1hr, the solution was assayed directly for PAL activity. For affinity purification of the U. maydis PAL enzyme, the isolated y-globulin was coupled to a HiTrap-NHS matrix according to the supplier's protocol. U. maydis PAL purified through the DEAE cellulose step was applied to the affinity column and eluted with 0.1 mM Na-citrate buffer, pH 3.0. 2.2.7 Western blot PAL proteins separated on a 7.5% native or SDS-PAGE gel were electrophoretically transferred to Westran polyvinylidene fluoride membrane(100 V, 1 hr, 4°C). Tris-glycine buffer (25 mM Tris-HCI, 190 mM glycine, pH 8.3) was used as transfer buffer for native PAGE gels. Methanol (10%) was included in the transfer buffer for SDS-PAGE gels. After electro-blotting, the Mr marker lane was cut off the membrane and stained with Coomassie Brilliant Blue R-250. The remaining blot was washed twice with TBS (100 mM 40 Tris-HCI, 150 mM NaCI, pH 7.5), blocked with 3% BSA in TBS-Tween buffer (0.1% Tween-20 in TBS) for 1 hr and probed with U. maydis PAL polyclonal antibodies for 2 hr at room temperature. After washing three times with TBS-Tween buffer, the blot was incubated (1 hr) with an alkaline phosphatase-conjugated goat anti-rabbit antibody. The blot was washed again and the secondary antibody detected with NBT/BCIP staining. 2.3 RESULTS 2.3.1 Purification of PAL The enzyme was most readily extracted from cultured U. maydis cells by grinding the cell mass with alumina powder. The degree of purification and the yield of PAL activity at each purification step are shown in Table 2.1. The elution profiles from the DEAE cellulose and Bio-Gel gel filtration columns showed one major PAL activity peak in each case (Fig. 2.1). Following chromatographic fractionation, the most active fractions were electrophoresed under non-denaturing conditions and the resulting gels were sliced and assayed. PAL was detected as a strongly trailing band of activity (Fig. 2.2), and electrophoresis in gels of different acrylamide concentrations (5-8%) showed that the mobility of the zone containing the highest PAL activity corresponded to a mass of 320+20 kDa (Fig. 2.3). Isoelectric focusing of the chromatographically purified PAL yielded one major protein band with a pi of 6.3 (Fig. 2.4A). No PAL isoforms were detected during either electrophoresis or chromatography, and the purified enzyme gave a negative reaction in the glycoprotein detection assay. 41 Table 2.1. Purification of PAL from U. maydis cells Purification Step Total Activity Protein Specific Activity Purification Recovery (pkat) (mg) (pkat/mg protein) (-fold) (%) Crude extract 17670 3442 5 1 100 Protamine Sulfate 14102 2174 6.5 1.3 80 30-60% (NH4)2S04 5552 328 17 3.4 31 DEAE Cellulose 5132 50 103 20.6 29 Bio-Gel A-0.5m 3331 2.1 1586 318 19 Preparative PAGE 530 0.090 5889 1178 3 42 E o -D < 1.2 x 0.9 --0.6 -• • - - Protein -A — Activity 0 0.3 •-1 i > M * j 20000 -- 16000 say) TO •• 12000 dpm -• 8000 > < -- 4000 a. * 0 10 19 28 37 Fraction Number 0.6 -o C O ~ 0.4 •• * 0.2 < a. 10 19 28 Fraction Number Fig. 2.1. Elution profiles of U. maydis PAL and protein after DEAE cellulose chromatography (upper) and Bio-Gel A-0.5m gel filtration (lower). 43 MIGRAT ION ! cm) 0 5 10 15 PAL A C T I V I T Y (DPMx10"3/assay) Fig. 2.2. Native PAGE analysis of purified PAL from U. maydis. After preparative native PAGE (6% gel), one lane was silver stained (left) and another was sliced into 0.5 cm sections and assayed for PAL activity (histogram, right). 44 0.3 0 I i i i i I 0 100 200 300 400 500 Molecular Weight (kDa) Fig. 2.3. Native molecular weight determination of U. maydis PAL by non-denaturing PAGE. The relative mobility of purified U. maydis PAL in 6, 7, 8, 9% polyacrylamide gels was compared to that of several high molecular weight standards (described in Materials and Methods). The molecular weight of PAL was determined by interpolation from the regression line. 45 A B Fig. 2.4. Isoelectric focusing PAGE (A) and SDS-PAGE (B) analysis of U. maydis PAL enzyme. Marker proteins (left lane) and enzyme (right lane) eluted from the native PAGE gel slice revealing highest PAL activity in Fig. 2.2, were electrophoresed, side by side, and then silver stained. Arrow indicates the location of PAL enzyme. 46 When the native PAGE gel slice corresponding to the zone of greatest enzyme activity was electro-eluted and subjected to SDS-PAGE, a single band of protein with a mass of 55 kDa was detected by Coomassie Blue staining. Silver staining, however, revealed three additional minor bands (84, 73, and 30 kDa) (Fig. 2.4B). 2.3.2 Antibody production and characterization Polyclonal antibodies raised against U. maydis PAL protein eluted from preparative non-denaturing PAGE gels produced a precipitin arc in immuno-double diffusion assays, with reaction to proteins from the crude extracts and from PAL-active fractions derived from ion-exchange and gel filtration separations (data not shown). The antibody titre was 12,000-fold as measured by dot immuno-blot assay. To test the effect of the U. maydis PAL antibodies on PAL enzyme activity, the U. maydis PAL enzyme was co-incubated with the y-globulin fraction of the U. maydis PAL antiserum. The PAL activity decreased linearly with increasing amounts of antibody, whereas the preimmune antibodies had no effect (Fig. 2.5). Immobilization of the anti-PAL y-globulin fraction to form an immunoaffinity matrix, and chromatography of partially purified U. maydis PAL preparations on this matrix, allowed recovery of low levels of PAL activity. The active fractions migrated as a single protein on non-denaturing PAGE and displayed the same mobility as conventionally purified PAL (data not shown). 47 a. 40 Y -•-PAL Antibody §| -m— Control Antibody 1 20 2 25 50 75 100 125 150 Antibody (ul) Fig. 2.5. Inhibition of U. maydis PAL activity by U. maydis PAL antibodies. The purified ^globulin fractions from U. maydis PAL antiserum and from preimmune serum (control antibody) were incubated with PAL enzyme for 1 hr at 25 °C and assayed for PAL activity. Activity is expressed as a percentage of a control not treated with antibodies. 48 2.3.3 Western blotting On Western blots, U. maydis PAL polyclonal antiserum detected a 320 kDa band in crude cell extracts, and in gel filtration-purified U. maydis PAL preparations, as well as an equivalent band in recombinant poplar PAL preparations (Fig. 2.6). When poplar PAL polyclonal antiserum (McKegney et al., 1996) was used as a probe, a weak band at 320 kDa was detected in purified U. maydis PAL (Fig. 2.6), but not in crude U. maydis cell extracts (data not shown). Activity assay of gel slices excised from the zone corresponding to the 320 kDa protein band detected on Western blots confirmed that the highest PAL activity was associated with this protein (data not shown). SDS-PAGE fractionation and Western blotting (Fig. 2.7) of U. maydis PAL preparations at different points in the purification revealed two strong protein bands (80 kDa and 52 kDa) and one weak band (160 kDa), with the 52 kDa protein predominating as the purification proceeded. 2.3.4 Stability When PAL enzyme in 0.01 M Na-phosphate buffer (pH 7.0) was heated for 10 min at 50 °C, cooled to room temperature and assayed at 30°C, no significant activity was lost (Fig. 2.8). Heating at 60°C or 70°C, however, resulted in activity losses of 30% and 100%, respectively (Fig. 2.8). When stored at 4°C, the enzyme retained 95% activity for a week, and 60% activity after one month. 49 Fig. 2.6. Western blot analysis of reciprocal cross-reactivity between recombinant poplar PAL, U. maydis PAL and their respective antisera. U. maydis PAL (1 or 10 u,g) from Bio-Gel filtration and purified recombinant poplar PAL (1 or 10 \ig) (McKegney et al., 1996) were separately electrophoresed in 7.5% native polyacrylamide gels and electro-blotted onto PVDF membrane. The blots were probed with either U. maydis PAL antiserum (1 \ig sample of U. maydis PAL and 10 u.g sample of poplar PAL), or poplar PAL antiserum (10 \ig sample of U. maydis PAL and 1 p.g sample of poplar PAL). After being probed with alkaline phosphatase-conjugated secondary antibodies, both the blots were developed with NBT/BCIP. Lanel: 1 ng U. maydis PAUU. maydis PAL antiserum^ :104. Lane 2: 10 \ig poplar PAUU. maydis PAL antiserum1:103. Lane 3: 10 \ig U. maydis PAL/poplar PAL antiserums 103. Lane 4: 1 \ig poplar PAL/poplar PAL antiserum1:104. Arrow indicates the location of PAL enzyme. 50 1 2 3 4 5 Fig. 2.7. Western blot analysis of U. maydis PAL subunit sizes at different stages of enzyme purification. Samples of PAL were taken from different purification stages, separated in 7.5% SDS polyacrylamide gels, and electro-blotted onto PVDF membrane. The molecular weight marker lane in the blot was stained with Coomassie Blue. Other lanes in the blot were probed with U. maydis PAL antibodies and alkaline phosphatase-conjugated secondary antibodies, followed by color development with NBT/BCIP. Lane 1: molecular weight markers. Lane 2: crude extract. Lane 3: 30-60% ammonium sulfate precipitate. Lane 4: Bio-Gel A-0.5m eluate. Lane 5: electro-eluate from a single band on a preparative native PAGE gel. 51 Fig. 2.8. Effect of temperature on U. maydis PAL activity. To measure temperature stability, the enzyme was preincubated at each different temperature for 10 min and cooled to room temperature for 30 min, and assayed at 30CC. 52 2.3.5 pH optimum The optimal pH for PAL activity was pH 8.8-9.2 (Fig. 2.9). The enzyme displayed 50% of maximum activity at pH 7.2. 2.3.6 Catalytic properties U. maydis PAL partially purified by DEAE cellulose ion-exchange chromatography displayed normal Michealis-Menten kinetics (Fig. 2.10). The apparent K„, value for L-phenylalanine was 1.05 mM, and the Vm a x was 51 pkat. mg"1. The enzyme displayed no detectable activity against L-tyrosine using either the spectrophotometric or radiometric assay methods. 2.3.7 Inhibitors and activators U. maydis PAL was readily inactivated by carbonyl reagents such as NaCN and NaBH4 (Table 2.2), as well as by the sulfhydryl reagent, p-chloromercuribenzoate. The enzyme was moderately sensitive to its reaction product, f-cinnamic acid (K\ = 0.41 mM), and to the product of cinnamic acid hydroxylation, p-coumaric acid, but the synthetic substrate analogue, 2-aminoindan-2-phosphonic acid (AIP) (Zon amd Amrhein, 1992), was a very effective inhibitor (Kj = 0.33 n.M). None of the metal ions tested produced any enhancement of PAL activity, but heavy metal ions generally inhibited the enzyme (Table 2.3). Treatment with EDTA was not 53 12000 w 9000 2- 6000 J 3000 K-Borate Buffer Oycine-NaOH Buffer 7.2 7.6 8 8.4 8.8 9.2 9.6 10 104 10.8 112 P H Fig. 2.9. Effect of pH on U. maydis PAL activity. To measure the optimum pH, the enzyme was preincubated at each different pH for 30 min and reacted with substrate for a subsequent 30 min at 30"C. 54 40 0 I i i i i • 0 500 1000 1500 2000 2500 [Substrate] (uM) 0.25 -1/[Substrate] Fig. 2.10. Kinetic analysis of partially purified U. maydis PAL. The most active fractions from DEAE cellulose ion-exchange chromatographed PAL were analyzed. Data were plotted on Michaelis-Menten (A) and Lineweaver-Burke (B) plot. 55 Table 2.2. Effect of chemical modification reagents and substrate analogues on U. maydis PAL activity Inhibitor Concentration Relative Activity (%) None - 100 NaCN 5 mM 0 NaBH4 5 mM 13 Phenylhydrazine HCI 5 mM 91 lodoacetate 5 mM 109 N-Ethylmaleimide 5 mM 32 yo-Chloromercuribenzoate 1 mM 11 /?-Mercaptoethanol 25 mM 40 AIP 0.1 u.M 55 10 u.M 1 f-Cinnamic acid 1 mM 75 10 mM 8 />Coumaric acid 10 mM 97 30 mM 0 56 Table 2.3. Effect of metal ions and chelators on U. maydis PAL activity Chemicals Concentration (mM) Relative Activity (%) None - 100 AgN03 5 3 Cd(CH3C02)2 10 38 CoCI2 10 62 CuCI 6 6 CuCI2 3 1 HgCI2 1 0.5 ZnCI2 10 54 8-Hydroxyquinoline 12.5 15 CaCI2, FeSCU, MgCI2, MnCI2, Na2Cr207, NaF, and EDTA at 10 mM concentrations did not reduce activity below 90% of control. 57 inhibitory, but the copper chelator, 8-hydroxyquinoline (12.5 mM), produced a moderate level of inhibition. PAL inhibition produced by Hg2+ could be partially reversed by treating the inhibited enzyme with p-mercaptoethanol, but 8-hydroxyquinoline was unable to reverse the inhibition produced by Cu+ or Cu 2 + ions. Although PAL requires a low level of a thiol protectant for long-term stability, high concentrations (>25 mM) of p-mercaptoethanol also inhibited the enzyme. 58 2.4 DISCUSSION Phenylalanine ammonia-lyase has been purified to varying degrees from numerous higher plant species but only from a limited number of fungi (Schomburg and Salzmann, 1990). It is a relatively low abundance protein and obtaining homogeneous preparations usually requires extensive fractionation of initial cell extracts. PAL is also prone to degradation during purification, and this lability can generate confusion concerning the native and subunit structure of the enzyme. The estimated molecular mass of native U. maydis PAL (320 kDa) is similar to most other known PAL enzymes, which typically range in mass from 300 to 340 kDa, although exceptions have been reported for PAL from Streptomyces (226 kDa) (Ernes and Vining, 1970), strawberry (266 kDa) (Given et al., 1988), and Altemaria (556 kDa) (Pridham and Woodhead, 1974). There is convincing evidence from studies of the heterologous expression of both fungal and plant PAL cDNAs that PAL is normally a homotetrameric protein consisting of four copies of the same gene product (Orum and Rasmussen, 1992; Appert et al., 1994). This implies that the subunit of the U. maydis enzyme should have a mass of 80 kDa and a protein of this size is prominent on Western blots of preparations obtained at various points during the purification procedure (Fig. 2.7). The Western blots show, however, that the original 80 kDa subunit population is accompanied by a strongly immunoreactive 52 kDa fragment throughout the normal purification process. The most highly purified active U. maydis PAL preparations (Fig. 2.2) consist of holoenzyme that yields almost exclusively this smaller fragment upon denaturation (Fig. 2.4.B, Fig. 2.7). 59 It is interesting to note that the undenatured enzyme appears to retain its native Mr even when the results of SDS-PAGE and Western blot analysis suggest that most of the subunits in the population have been cleaved to yield a discrete, substantially smaller, polypeptide fragment. This behavior, which has also been observed in other systems (Bolwell et al., 1986; Dubery and Smit, 1994), implies that the cleavage fragments remain firmly bound within the tetrameric structure of PAL. Heterotetrameric quaternary structures for PAL have been reported from Rhizoctonia (Kalghatgi and Subba Rao, 1975) and sunflower (Jorrin et al., 1988), but partial degradation of the enzyme cannot be ruled out in either case. The U. maydis PAL antiserum has a high affinity for both the native fungal enzyme and its denatured subunits. The U. maydis PAL antiserum was also able to recognize a higher plant PAL protein, although the reaction was far weaker than with the homologous protein. The reciprocal cross-reaction between poplar PAL antiserum and U. maydis PAL protein (Fig. 2.6) was similarly detectable but weak. While antisera have been raised to several plant and fungal PAL proteins (e.g. alfalfa (Jorrin and Dixon, 1990), bean (Bolwell era/., 1985), and Mycosphaeria maculans (Dahiya, 1993)), I am unaware of any other reports on the degree of cross-reactivity between plant and fungal PAL. The cross-reactivity observed here is consistent with the presence of some short stretches of highly conserved sequence within plant and fungal PAL genes (Taylor et al., 1990), but the weakness of the cross-reaction also emphasizes the extent to which the structures of the Ustilago and higher plant PAL proteins may have diverged. 60 Many of the physico-chemical properties of U. maydis PAL are typical of this enzyme from other sources (e.g. Mr, pH optimum, sensitivity to carbonyl reagents, requirement for thiol protectants). Other properties are more unusual, including the temperature stability, a relatively high apparent K m for phenylalanine, and a pi of 6.3. PAL proteins from other species have been reported to display apparent K m values ranging from 11 uM to 450 uM L-phenylalanine, while the reported pi values range from 2.5 (Neumann et al., 1991) to 5.75 (Campbell and Ellis, 1992). PAL preparations from some sources, including sweet potato (Minamikawa and Uritani, 1965), tobacco (O'Neal and Keller, 1970), and yeast (Parkhurst and Hodgins, 1972), are strongly inhibited by the reaction product, f-cinnamic acid, but cinnamic acid has comparatively little effect on the activity of the U. maydis enzyme. In this, the Ustilago PAL resembles the enzyme from Streptomyces (Ernes and Vining, 1970), alfalfa (Jorrin and Dixon, 1990), pine (Campbell and Ellis, 1992), and tomato (Bernard and Ellis, 1991). Like most known PALs, the Ustilago enzyme is sensitive to heavy metal ions and displays no metal ion requirement for catalytic activity (Table 2.3). Again, there is considerable interspecific variability in these responses, since Hg2+ completely inhibited the U. maydis PAL enzyme at 0.5 mM, whereas sunflower PAL was less strongly inhibited, retaining some activity even in 2 mM Hg2+ (Camm and Towers, 1973). Similarly, Cu 2 + (1mM), was a potent inactivator of the U. maydis PAL but had no effect on the sweet potato enzyme (Minamikawa and Uritani, 1965). The significance, if any, of these patterns will become apparent only when more is learned about the detailed structure of PAL proteins and their active site(s). 61 CHAPTER THREE Cloning and Characterization of the PAL Gene from Ustilago maydis: Relationships with Other Phenylalanine Ammonia-lyases and with Bacterial Histidine Ammonia-lyase. 3.1 INTRODUCTION Since its discovery by Koukol and Conn (1961), L-phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) has been studied extensively both from the perspective of its biochemistry and its molecular biology. Plant PAL activity levels respond to an array of stresses such as wounding, chemicals, microbial infection, and UV light (Jones, 1984). By catalyzing the conversion of L-phenylalanine to /-cinnamic acid, the enzyme regulates the biosynthesis of a huge class of phenylpropane skeleton-based natural products (Hahlbrock and Scheel, 1989). Partial or full-length genes encoding PAL have been isolated from a number of plant species, mostly from cultivated crops but including trees and Arabidopsis. PAL is encoded by a small gene family in most plants except in potato, where PAL is encoded by 40-50 genes (Joos and Hahlbrok, 1992), and in loblolly pine (Pinus taeda), where PAL is reported to be encoded by only one gene (Whetten and Sederoff, 1992). In general, PAL coding sequences range from 700 to 725 amino acids in length, with some exceptions such as 682 amino acids in poplar PAL (Osakabe et al., 1995), 694 in Arabidopsis PAL (Wanner et al., 1995), and 752 in loblolly pine PAL (Zhang and Chiang, GenBank accession no. U39792). The fungal PAL genes sequenced to date contain five or six introns (Anson et al., 1987; Vaslet et al., 1988), in 62 contrast to plant PAL sequences which generally contain only one intron. Two exceptions are the Arabidopsis PAL gene, which has two introns (Wanner et al., 1995), and a Pinus banksiana PAL gene which appears to lack introns (Campbell, 1991; S. Butland, personal communication). The strict sequence conservation in the second exon of PAL genes among different plant species implies that important catalytic sites of the enzyme may be encoded in this region (Cramer et al., 1989; Joos and Halbrock, 1992). While PAL has been well-studied in plants, relatively little is known about PAL in fungi. The enzyme has been purified from a limited number of fungi (Schomburg and Salzmann, 1990), and fungal PAL genes have only been isolated from the two closely related red yeasts, Rhodosporidium toruloides and Rhodotorula rubra (Anson et al., 1987; Vaslet et al., 1988). The role of PAL in fungi is an area which is largely unexplored. Investigation of the structure, function, and regulation of fungal PAL will enable us to better understand the role PAL plays in fungal biology and may help us leam more about the mechanisms by which PAL is controlled in different eukaryotic systems such as plants and fungi. The phytopathogenic fungus Ustilago maydis provides a good model system in which address the above question. This species has been used to study many aspects of eukaryotic genetics, including recombination mechanisms (Holliday et al., 1974), nitrogen metabolism (Lewis and Fincham, 1970), and developmental regulation (Froeliger and Kronstad, 1990). Molecular genetic approaches are also possible, with the development of such molecular tools as efficient transformation (Wang et al., 1988), autonomously replicating vectors (Tsukuda et al., 1988), genes for selection markers 63 (Wang et al., 1988; Banks and Taylor, 1988), and gene disruption (Kronstad et al., 1989). The availability of U. maydis PAL protein data and anti-PAL antibodies (chapter 2) provided a unique opportunity to pursue the molecular genetic studies of PAL in a versatile fungal species largely unrelated to the red yeasts. Histidine ammonia-lyase (HAL, EC 4.3.1.3) and PAL are the sole members of the dehydroalanine class of ammonia-lyases. HAL catalyzes the nonoxidative deamination of L-histidine to frans-urocanic acid and ammonia. This is the first step in the catabolism of L-histidine, which is eventually metabolized to L-glutamic acid. HAL occurs generally in bacteria and animals but may occur universally, since HAL activity has been detected in a few fungi (Hollman and Dekker, 1971; Polkinghorne and Hynes, 1975) and plants (Ruis and Kindl, 1970 and 1971; Kamel and Maksoud, 1978). In bacteria, HAL allows utilization of histidine as a carbon and/or nitrogen source. In mammals, HAL is under complex regulation that controls activity in a developmental, hormonal, and tissue-specific manner (Feigelson, 1973; Lamartiniere, 1979; Armstrong and Feigelson, 1980). For both HAL and PAL there is strong indirect evidence that the enzyme active site contains a catalytically essential dehydroalanine (DHA) residue (Hanson and Havir, 1970; Hodgins, 1971; Consevage and Phillips, 1985). Recently, the precursor of the dehydroalanine residue in HAL and PAL has been identified as serine located at position 143 in Pseudomonas putida HAL (Hernandez etal., 1993; Hernandez and Phillips, 1994; Langer et al., 1994), 254 in rat HAL (Taylor and Mclnnes, 1994), and 202 in parsley (Schuster and Retey, 1994) and in poplar PAL (McKegney et al., 1996). Substantial amino acid sequence conservation between HAL (from rat, Bacillus subtilus, and P. 64 putida) and PAL (from yeast, parsley, and kidney bean) was noted by Taylor et al. (1990). On the basis of functional similarity, the presence of DHA at the active site, and the observed overall sequence conservation, these researchers proposed that the present-day genes for HAL and PAL could have evolved from a common ancestral ammonia-lyase gene. Since this proposal has been made, more HAL and PAL genes have been cloned from other sources. The relatedness of the two enzymes has been inferred from recent studies of their mechanism of action (Schuster and Retey, 1995) and the comparison of some coding sequences (Wu et al., 1992), but many questions about these DHA ammonia-lyases remain unresolved, including the mechanism of active site formation and the functional significance of their multimeric structure. At this point, there have been no X-ray crystallographic analyses reported that might provide insight into these issues. However, a better understanding of the structural features common to both HAL and PAL should provide useful insights into the way an active DHA ammonia-lyase is formed. This chapter describes the isolation and sequencing analysis of a U. maydis genomic clone which contains a gene encoding PAL, and a comparative analysis of its protein sequence with some of the published PAL and HAL protein sequences. Immunological evidence regarding the relatedness of the two ammonia-lyases is also presented. 65 3.2 MATERIALS AND METHODS 3.2.1 Microorganisms and media Escherichia coli strains employed in this study are listed in Table 3.1. E. coli strains were grown in LB or TB medium with appropriate antibiotics as described (Sambrook et al., 1989) and Ustilago maydis 518 strain was grown on potato dextrose broth or agar (PDB or PDA, Difco) and on complete medium (CM) described by Holliday (1974). Rhodosporidium toruloides UBC 75-0941 was grown in malt extract agar (Difco). 3.2.2 Materials Restriction and DNA modifying enzymes were purchased from Bethesda Research Laboratories (BRL), Boehringer Mannheim and Pharmacia. The 1 Kb DNA Ladder, RNA size markers and agarose were obtained from GIBCOBRL. [a-32P]dATP was from Amersham. Zeta-Probe blotting membranes for Southern and Northern blot analysis, Bio-Gel A-0.5m agarose beads, alkaline phosphatase-conjugated goat anti-rabbit antibody, SDS-PAGE molecular markers, and BCIP (5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt)/NBT (p-nitro blue tetrazolium chloride) were purchased from Bio-Rad (Mississauga, Canada). Westran PVDF (polyvinylidines difluoride) membrane was purchased from Schleicher & Schuell (Keene, U.S.A.). Freund's adjuvant and other chemicals were purchased from Sigma. Degenerate oligonucleotide primers were synthesized by Nucleic Acids and Protein Services (NAPS) unit, Biotechnology Lab., 66 Table 3.1. Escherichia coli strains used in this study Strain Genotype DH5a endA\, hsdRM(rk~, mk+), supEAA, ff?A-1, recAl, gyrA96, re/,41, J/acL/169 (<j>80d/acZAM15) DH10B F' mcrA A(/r7nrA7SdRMS-mcrBC)«))80d/acZAM15), AlacX7A, deoR, recAl, araD139, A(ara, leu)7697, galU, galK, XT, rpsL, endAl, nupG HB101 supEAA, AjsdS2(rB~, m B l , rec>413, ara-14, proA2, lacY\, ga/K2 rpsL20, xyl-5, rrrtlA SOLR e14 ~ (mcrA), A(mcrCB-hsdSMR-mrr)'\7'\, sbcC, recB, recJ, umuC:Jn5(kari ), uvrC, lac, gyrA96, reM1, fliM, er»dA1, AR, [F', proAB, /ac/qZAM15], Sir (non-suppressing) XL1-Blue endA-\, /jsa7?17(rk~, mk+), supEAA, thn, lambda", rec>A1, gyrA96, relA\, lac', [F', proAB, lacPTAmb, TnlOtfef*)] 67 University of British Columbia and specific primers were synthesized using an Oligo-1000M DNA Synthesizer (Beckman). 3.2.3 Nucleic acids manipulation Protocols used for recombinant DNA manipulations and for small scale, boiling-lysis, plasmid preparations are from Sambrook et al. (1989). DNA mini-preps were done by the method of Zhou er al. (1990). Restriction digests and ligations were carried out according to manufacturers' instructions using commercially supplied enzymes and buffers. pUC19 vector and DH5a competent E. coli cells were used for subcloning. Genomic DNA of U. maydis and R. toruloides was isolated as described by Wang et al. (1988). U. maydis RNA was isolated using the hot phenol method as described in Ausubel et al. (1995). Concentration of nucleic acids was determined by measuring the absorbance at 260 nm using a GeneQuant nucleic acids quantifier (Pharmacia). 3.2.4 cDNA library screening A XZAPII cDNA expression library (Gold et al., 1994a) constructed from poly(A)+ RNA from U. maydis cells grown on mating media was used as the source for PAL cDNA clone screening. E. coli XL-1 blue cells (Stratagene) were infected with the phage library, lacZ fusion protein expression was induced with isopropyl-yS-D-thiogalactopyranoside (IPTG), and positive clones were selected by immunoscreening with antibodies specific for U. maydis PAL. Immunoscreening was carried out with a 68 p/'coBlue immunoscreening kit (Stratagene) using alkaline phosphatase-conjugated goat anti-rabbit antibodies with BCIP and NBT as substrates for color detection. Putative positive plaques were isolated through several rounds of screening and processed to homogeneity. The plaques giving a positive signal were excised in vivo with the EXASSIST helper phage (Stratagene) and rescued to produce the pBluescript SK-plasmid using the E. coli strain, SOLR (Stratagene) which prevents coinfection of the helper phage, according to the Stratagene EXASSIST/SOLR system protocol. Transformed cells were stored as glycerol stocks at -70 °C. 3.2.5 Design of oligonucleotide primers The DNA coding sequences and amino acid sequences for known PAL genes were compared using PC/Gene software (IntelliGenetics) and assessed to identify consensus regions which could be used as PCR primers. The sequences employed for comparison were four plant PAL sequences: Ipomea batatas (Genbank/EMBL accession M29232), Oryza sativa (accession X16090), Phaseolus vulgaris (accession M11937), and Petroselinum crispum (Lois et al., 1989), and two yeast PAL sequences: Rhodotorula mbra (accession X13094) and Rhodosporidium toruloides (accession M28261). Degenerate primers (PAL1, 2, 5, 6, and 7) and yeast PAL-specific primers (RTPAL3 and 5) were synthesized based on consensus region sequences (Fig. 3.1). 5' EcoRI or 3' Xbal restriction sites were incorporated in the primers to allow unidirectional cloning of amplified products. 69 Generic PAL Gene (coding region) 5' 1 100 200 I 300 400 500 1 Amino acids 600 3' 700 1 I t active site PAL5 PAL7 1 i i r known intron site in plants or fungi => PAL1 PAL2 RTPAL5 RTPAL3 PAL6 PAL1 5'-CGGAATTC rACIGIACHTAirCGllCCICArAGlTGGrTCIT -3' EcoRI PAL2 5'-GCTCTAGA TGITClTCIGCirGCirATirTClTGIACrAGlTG -3' Xbal PAL5 5'-CGGAATTC TACGGrTClGTCACIACrTClGGITTrTClGG -3' EcoRI PAL6 5'-GCTCTAGA TTIACrGAlTCrCTITGrGAlTTrAGlTGrCTITG -3' Xbal PAL7 5'-CGGAATTC ATCrATlCGGCGTCGGGGGArCTirCTIT 3' EcoRI RTPAL3 5'- GCTCTAGA CGCAAGGGGTAGCGGTCCT -3' Xbal RTPAL5 5'- CGGAATTC CACTCGGCTGTCCGCCTCGT -3' EcoRI Fig. 3.1. Schematic diagram indicating generic structure of a PAL gene and locations of primers (=>) used in PCR to amplify parts of the U. maydis PAL gene. Degeneracy of primers is indicated by square brackets and the inosine nucleotide is indicated as I. 70 3.2.6 PCR amplification of a putative U. maydis PAL gene fragment U. maydis genomic DNA and AZAPII-cDNA from the cDNA library were prepared using the yeast DNA isolation method and the plate lysate method, respectively, as described in Current Protocols in Molecular Biology (Ausubel et al., 1995). PCR reactions (50 uJ) contained 200 ng of U. maydis genomic DNA or 20 ng of XZAPII-cDNA as template, 40 pM PAL primer pairs, 50 u.M dNTPs, 1X reaction buffer (10mM Tris-HCI pH 8.0, 1.5 mM MgCI2, 50 mM KG), and 1 unit Taq polymerase (Appligene). Each reaction was overlayed with 1 drop of mineral oil. The combinations of primers and templates for PCR reactions are listed in Table 3.2. PCR reactions were incubated in a Techne PHC2 thermal cycler. The thermal cycling conditions were as follows: initial denaturation, 94°C/4 min; followed by 35 cycles of denaturation, 94°C/1 min; annealing, 55°C/1 min; and primer extension, 72°C/2 min. The reaction product (30 u.l) was analyzed by electrophoresis on a 1% agarose gel in Tris-acetate EDTA (TAE) buffer including ethidium bromide, visualized under UV light, and documented with an Image Analyzer (IS-500 Digital Imaging System, Alpha Innotech Co.). After identification of the desired size band from amplification products, this band was excised, purified using the GeneCleanll (Bio101) kit, and subcloned for further analysis. 3.2.7 Cloning of putative U. maydis PAL PCR product The purified PCR amplified product was digested with EcoRI and Xbal, ligated with pUC19 vector digested with the same enzymes, and transformed into DH5a E. coli cells. Transformant colonies were screened using blueAvhite selection based on the insertional 71 inactivation of p-galactosidase. Twenty white colonies were picked for plasmid DNA minipreps (Zhou et al., 1990), digested with EcoRI and Xoal, and subjected to electrophressis on 1% agarose gels to verify the insert presence and size. The identities of transformants with an insert size corresponding to the amplified PAL DNA fragment were confirmed by Southern blot analysis of plasmid DNA from the selected clone using the PCR-amplified PAL fragment as a probe. The amplified putative U. maydis PAL fragment DNAs in the confirmed clones were sequenced and their sequences were compared with other known PAL sequences. A fragment showing substantial sequence homology with yeast PALs was used as a probe to screen a U. maydis genomic-cosmid library. 3.2.8 DNA sequencing Double-stranded plasmid DNA or cosmid DNA from selected clones was prepared following a mini alkaline lysis/PEG precipitation procedure (Ausubel et al., 1995). Both strands of the insert were sequenced with M13 universal and/or synthetic oligonucleotide primers as needed to extend the sequence. Sequencing reactions were carried out in a DNA Thermal Cycler (Perkin Elmer Cetus) using ABI AmpliTaq dye termination cycle sequencing chemistry according to manufacturer's instructions (Applied Biosystems) and nucleotide sequences were analyzed on Applied Biosystems' ABI 373 DNA sequencer (NAPS unit) or ABI 310 capillary DNA sequencer (at the former Pacific Agriculture Research Centre, Agriculture and Agri-Food Canada, Vancouver Station). 3.2.9 Sequence comparison and analysis 72 Database searches for sequence homology and comparisons of nucleotides and amino acids were performed on sequences from Genbank, EMBL, PDB, PID, PIR, and SWISS-PROT using BLAST (Altschul et el., 1990). Nucleotide and amino acid sequence analysis of U. maydis PAL was done using PC/Gene software. 3.2.10 Genomic-cosmid library screening A cosmid library had been constructed earlier (Barrett, 1992) in the U. maydis vector pJW42, using total genomic DNA isolated from U. maydis strain 518. The total DNA was partially digested with Saw3AI to produce 30 to 40 kb fragments and these fragments were ligated into the BamHI site of pJW42. After being packaged in vitro using the Gigapack packaging extract (Stratagene), ligated DNA was transfected into E. coli DH5ct strain. Approximately 106 individual transfectants were randomly pooled from transfection plates (LB/ampicillin (50 ng/ml) medium), cultured for six hours in LB/ampicillin medium, and harvested for DNA preparation (Ish-Horowicz and Burke, 1981). DNA from these nine divided pools was used as a source of genomic-cosmid library for U. maydis genomic PAL screening. To identify the cosmid DNA pool containing a PAL gene, PCR was employed, using nested primers (UMPAL2 and UMPAL3), which had been designed on the basis of sequence information from the PCR-amplified putative PAL DNA fragment. Cosmid DNA from the pools giving a PCR amplification product with the nested primers was transformed into E. coli HB101 or DH10B strain by electroporation, using a Bio-Rad Gene Pulser according to the supplier's manual, and transformants were selected on LB/ampicillin plates. The selected transformants were screened 73 according to Amersham's colony-lift hybridization manual using the PCR-amplified putative U. maydis PAL DNA fragment cloned above as a probe. After growing on antibiotic selective medium plates, bacterial DNA was transferred and fixed onto nylon membrane (Hybond N+, Amersham). Membranes were prehybridized for 1 hour at 68°C and hybridized with the DNA probe overnight at 68°C. After washing with 2XSSC (10 min, twice) at room temperature and 0.1X SSC/0.1% SDS at 68°C (30 min, twice), the membranes were exposed to Kodak X-OMAT film. Colonies giving positive signals on autoradiograms from duplicate membranes were picked and purified to homogeneity through two more rounds of screening. Cosmid DNA from these selected clones was subjected to further analysis by PCR with UMPAL2/UMPAL3 primers to confirm the presence of target template DNA and the confirmed cosmid DNA was used for sequencing analysis. 3.2.11 Southern hybridization U. maydis genomic DNA was digested overnight at 37°C with various restriction enzymes, fractionated in a 0.7 or 1% agarose gel, denatured in 0.25M HCI for 10 min, and transferred to a Zeta-Probe GT blotting membrane using 0.4M NaOH solution. The membrane was prehybridized at 65°C for 1 hour in 10 ml of hybridization solution (7% SDS, 0.25M Na2HP04, pH 7.2) and transferred to new hybridization solution. After addition of a DNA probe which was labeled with [<x-32P]dATP using the Random Primers DNA Labeling System (GIBCOBRL), the membrane was incubated overnight at 65°C. The membrane then washed twice for 30 min each at 65°C in 5% SDS/20 mM Na2HP04, 74 pH 7.2, followed by washing in the same manner in 1% SDS/20 mM Na2HP04, pH 7.2, and subjected to autoradiography using Kodak X-OMAT AR film. 3.2.12 Total RNA isolation and Northern hybridization Cells of U. maydis grown in complete medium were harvested 16 hr after inoculation, washed two times with sterile DEPC-treated distilled water, and frozen in liquid nitrogen. Total RNA was isolated using the hot phenol extraction method as described by Ausubel et al. (1995). Total RNA was fractionated on a denaturing 1% agarose gel (Sambrook et al., 1989) and transferred to Zeta-Probe GT membrane using 10X SSC. The membrane was incubated in prehybridization solution (50% formamide, 0.12M Na2HP04, 0.25M NaCI, and 7% SDS) for 10 min at 42°C and hybridized overnight with a random-primed radiolabeled probe in the same conditions. Following a brief rinse in 2X SSC, the membrane was washed successively in 2X SSPE/0.1% SDS, 1X SSPE/0.5% SDS, and 0.3X SSPE/1% SDS solution at 55°C for 30 min each. A 0.24- to 9.5-kb RNA ladder was used to estimate RNA size. Autoradiography was performed in the same manner as for the Southern blot. 3.2.13 N-terminal and internal peptide sequencing of U. maydis PAL The purified U. maydis PAL (chapter 2) was run in 7.5% SDS-PAGE gels (Laemmli, 1970) and subjected to electroblotting onto Immobilon P^-PVDF membrane (Millipore) using CAPS buffer. After staining with Coomassie blue, the appropriate 80 kD and 52 kD protein bands were excised for N-terminal and internal sequencing analysis, respectively. 75 Protein sequencing analysis by automated Edman degradation was performed on an Applied Biosystems 470A gas phase sequencer with on-line PTH-HA analysis at the NAPS unit. 3.2.14 Enzymes and antibodies Partially purified Pseudomonas fluorescens HAL, R. glutinis PAL, and potato PAL were purchased from Sigma. P. fluorescens HAL was further purified from the commercial enzyme by 7.5% native PAGE gel separation and polyclonal antibodies were raised in rabbits. U. maydis PAL was purified by (NH4)2S04 precipitation, DEAE ion-exchange chromatography and Bio-Gel A-0.5m filtration (described in chapter 2). Poplar PAL was purified from baculovirus-expressed proteins (Mckegney et al., 1996) by Pharmacia FPLC Mono-Q column. Polyclonal antibodies to U. maydis and poplar PAL were raised in New Zealand White rabbits. Anti-alfalfa PAL and anti-P. putida HAL polyclonal antibodies were generously provided by R.A. Dixon (The Samuel Robert Noble Foundation, Ardmore, USA) and AT. Phillips (Pennsylvania State University, University Park, USA), respectively. 3.2.15 Western blots and enzyme Inhibition The same amounts of protein of each PAL and HAL enzyme were electrophoresed on a 7.5% native PAGE gel, electroblotted onto PVDF membrane and probed with anti-PAL or anti-HAL antiserum. After washing, the membrane was reacted with alkaline phosphatase-conjugated secondary antibodies and immune complexes were detected with NBT/BCIP solution (Young and Davis, 1983). For enzyme inhibition by antiserum, 76 HAL and PAL were incubated with anti-HAL and anti-PAL antisera for 1hr at room temperature and the enzyme-antiserum reaction mixtures were assayed for HAL and PAL activity. Pre-immune sera were used as controls. 3.2.16 Assay of enzyme activity HAL assays were done by the spectrophotometric method with L-histidine as a substrate (Rechler and Tabor, 1969) and PAL assays were done by the radiometric method with L - [l/-14C]-phenylalanine as a substrate (Bernards and Ellis, 1991). Protein concentrations were determined using the Bio-Rad dye-binding reagent with microtiter plates, based on the Bradford method (Bradford, 1976). 3.3 RESULTS 3.3.1 PCR amplification of a U. maydis PAL sequence from genomic DNA Initial attempts at screening available U. maydis cDNA libraries with either U. maydis PAL polyclonal antibodies (chapter 2), or with a yeast R. toruloides PAL DNA fragment (PCR amplified from R. toruloides genomic DNA with primer RTPAL3 and RTPAL5 in this study), or with a Pinus banksiana PAL cDNA clone (Lam, 1995) as probe were unsuccessful. This failure may have been due to the low abundance of the PAL message in the mRNA pools from which the libraries were made or to low nucleotide sequence homology between the target U. maydis cDNA in the cDNA library and the 77 heterologous probes. This led me to choose a PCR approach, generating primers (Fig. 3.1) based on conserved amino acid sequences among several PAL proteins. Among the combinations of primers and template DNA tested (Table 3.2), only the PAL7 and RTPAL3 primer combination with genomic DNA as template amplified a fragment of the expected size (about 0.45 kb) (Fig. 3.2). This DNA fragment was isolated, subcloned into pUC19, transformed into DH5a E. coli cells, and sequenced. A BLAST database search showed that the 0.45 kb-sequence has homology with most known PAL genes, especially with yeast PALs (40% amino acid identity). Therefore, the PCR-amplified DNA was considered a putative U. maydis PAL DNA fragment and was used to screen a genomic-cosmid library. 3.3.2 Isolation and sequencing of PAL genomic-cosmid clones Based on the sequence of the above 0.45 kb PCR product, two sequence-specific primers (UMPAL2 5'-ACGCAGCGTTCCGTTGTCTTC-3') and UMPAL3 (5'-AGCTACGTAGCCGGTGCGCTT-3') were synthesized and used for initial screening of nine genomic-cosmid pools. A 0.43 kb fragment was amplified from three pools (pool 1, 7, and 8) out of nine pools (Fig. 3.3). After transformation of DNAs of these three pools into HB101 or DH10B cells, PAL genomic clones were isolated from the transformants by colony-lift hybridization, using the 0.45 kb DNA as probe. One clone was selected for sequencing after rescreening of the above isolated PAL clones by PCR with the UMPAL2/UMPAL3 primers. DNA sequencing of both strands of this clone was performed using synthetic oligonucleotide primers designed from the DNA sequences resulting from each sequencing run (Fig. 3.4). 78 Table 3.2. PCR primer combinations tested for possible amplification of PAL DNA fragments from U. maydis and predicted size of the amplified products Primer combination Predicted size (bp) cDNA template Genomic DNA template PAL1-PAL2 400 > 400 PAL1-PAL6 420 > 420 PAL5-PAL2 1100 >1100 PAL5-PAL6 1120 £ 1 1 2 0 PAL5-RTPAL3 700 > 700 PAL7-PAL2 840 > 840 PAL7-PAL6 860 > 860 PAL7-RTPAL3 450 450 RTPAL5-PAL2 950 > 950 RTPAL5-PAL6 970 > 970 RTPAL5-RTPAL3 550 550 PALI-Ml3 1050 PAL 5-Ml 3 1740 PAL7-M13 1480 RTPAL5-M13 1590 79 0.45 Fig. 3.2. PCR amplification of putative PAL DNA fragment from U. maydis genomic DNA using various PAL primer combinations. The amplified products were separated electrophoretically in a 1% agarose gel and visualized under UV following ethidium bromide staining. Lane 1: 1kb DNA ladder, Lane 2: primer PAL7/RTPAL3, Lane 3: primer PAL7/PAL2, Lane 4: primer PAL5/RTPAL3, Lane 5: primer PAL5/PAL2, Lane 6: primer PAL1/PAL2, and Lane 7: primer RTPAL5/RTPAL3. 80 1 2 3 4 5 6 7 8 9 10 Fig. 3.3. PCR screening of U. maydis genomic-cosmid library pools using UMPAL3 and UMPAL2 primers. PCR products were separated electrophoretically in a 1% agarose gel and visualized under UV following ethidium bromide staining. Lane 1: 1kb DNA ladder, Lane 2: genomic-cosmid pool 1, Lane 3: pool 2, Lane 4: pool 3, Lane 5: pool 4, Lane 6: pool 5, Lane 7: pool 6, Lane 8: pool 7, Lane 9: pool 8, Lane 10: pool 9. 81 300 bp "ATG" "TAA" PAL translation start PAL PAL termination 1 5' 3' < < < < < < < < > > > > > > > > Fig. 3.4. Strategy employed in the nucleotide sequencing of the U. maydis PAL genomic-cosmid clone. Arrows indicate the direction and extent of DNA that was sequenced. The dotted line represents total sequenced nucleotides of the PAL genomic clone. The thick black line above the dotted line indicates the 2172 bp open reading frame encoding PAL. The sites for the presumed PAL translation initiation and termination are indicated. The box indicates the location of the PCR-amplified 0.45 kb product from Fig. 3.2. 82 3.3.3 Nucleotide sequence and deduced amino acid sequence of U. maydis PAL A total of 3047 bp nucleotide sequence of a genomic PAL clone was determined (Fig. 3.5). This contains 495 bp of 5' untranslated sequence, a 2172 bp open reading frame encoding 724 amino acids, and 380 bp of 3' untranslated sequence. A BLAST database search for homology of the 2172 bp sequence with known genes showed that this sequence has highest homology with known PAL genes (Table 3.3). A putative 'TATA box' is present 349 bp upstream from the start codon. However, typical eukaryotic promoter sequences are not found in the usual locations upstream from the U. maydis PAL gene start codon. The GC content of the coding sequence is 58%. No introns are located in the U. maydis PAL sequence. The 3' untranslated region does not possess any of the consensus transcriptional termination signals of the higher eukaryotes (AATAAA) or of Neurospora (TGTCGA). Conceptional translation of the 2172 nucleotide sequence of this U. maydis PAL gene is presented in Fig. 3.6. The molecular weight and isoelectric point of the translated 724 amino acid protein are estimated as 79277 and 6.3, respectively, which are in good agreement with the previously estimated subunit size and isoelectric point of purified PAL (molecular weight 80000 and pi 6.3 in chapter 2). The known enzyme active site serine residue is found at position 206 within the strictly conserved motif SGDL found in PAL genes (Schuster and Retey, 1994) (Fig. 3.6). An internal peptide fragment sequence from the purified U. maydis PAL matches positions 384-391 (underlined) of the deduced amino acid sequence (Fig. 3.6). Interestingly, /V-glycosylation sites, a cAMP- or GMP-dependent protein kinase phosphorylation site, and a ATP/GTP-binding site motif 83 GGTGCTCCCCAACAAATGGCGCGCTTTTTTCGGTAGCATGCAGGATAATCTGTTCATCAC - 4 3 6 TGTGAGGGTTCACGTTCGTAATTAAACAAAGCGCACATTLCCTGTTTGGATGTCATCGGA - 3 7 6 TATTCCGCGACAACTCGGTA TATTAI rAGTGTAGTTTGACAGAGGGAGTGGACGCGGCTG - 3 1 6 AGATGGGACCGTTCCGTGTCAGGAGAGTGGACAACGCATTGCGCGGAATGAAGTCAGAAT - 2 5 6 CGATGCATCAATGATTCACGATTGTTGCTCTGACGATCGGCTCGCCCGTTCCGTTCGCGG - 1 9 6 T G C G C A T C C T G A T T G C C A G A T A G C C A G A G A C G T G G A G C C T G A A G G T G A C T A T A G T A T G G G - 1 3 6 A C A G C A A T C C T A G C C G A C T T T C T C A C C T C C T A T C C G C C C A T A T C T G C G T G C C G T G C C T C T - 7 6 T C G A T C G T C T C T A C A C G A C C A T A A C A G C T G T C C T C T C G C G T C C A T A C C G T T C C T C T T C C C - 1 6 A C C G C A T C T G G C A T C A T G G C T C C A A C C G C A G A C G T G C T C C C T C C C G T C G A G G C A T C C A C G 45 M A P T A D V L P P V E A S T 15 C G T C C A G G C T T G C T C G T C C A G C C T T C G G A T A C C A A A C T T C G C A A A G C A T C G T C C T T C C G A 105 R P G L L V Q P S D T K L R K A S S F R 35 A C C G A G C A G G T C G T T A T C G A C G G C T A C A A T C T C A A G A T C C A G G G T C T C G T C G C T T C C G C T 165 T E Q V V I D G Y N L K I Q G L V A S A 55 C G A T A C G G T C A C G T T A C C C G T C C T C G A C C C T C C G C T G A G A C G C G A A A G C G T A T T G A T G A C 2 2 5 R Y G H V T R P R P S A E T R K R I D D 75 T C G G T C C A G T C C T T A A T C G C C A A G C T C G A C G G T G G C G A G T C A A T C T A C G G C A T C A A C A C G 2 8 5 S V Q S L I A K L D G G E S I Y G I N T 95 G G G T T C G G T G G G T C C G C C G A C T C G A G G A C C G C C A A C A C A C G T G C G C T T C A G C T G G C C T T G 345 G F G G S A D S R T A N T R A L Q L A L 115 C T C C A G A T G C A G C A G T G T G G C G T G C T C C C C G T G C C A T C C A C A T T C C C C A C G G G C G A A C C C 4 0 5 L Q M Q Q C G V L P V P S T F P T G E P 135 A G C T C G G C A C C C T T T G C A C T C C C T T T G A C G G A C A C A G A G T C T T C A C T G A T C A T G C C G G A G 4 6 5 S S A P F A L P L T D T E S S L I M P E 155 G C A T G G G T A A G G G G T G C C A 4 C G T G G T T A G G C T C A G C T C T C T G A T G C G C G G T C A T T C G G G T 525 A W V R G A I V V R L S S L M R G H S G 175 G T G C G T T G G G A G G T G C T C G A C A A G A T G C A G A A G C T T T T C C T C C A G A A C A A C G T C A C T C C A 585 V R W E V L D K M Q K L F L Q N N V T P 195 GTCGTACCAGTCAGGTCGAGTATCTCGGCCA6TGGTGATCTTAGCCCACTTAGCTACGTA 645 V V P V R S S I S A S G D L S P L S Y V 2 1 5 GCCGGTGCGCTTGCCGGTCAGCGTGGCATCTACTGCTTTGTCACCGACGGCCGTGGTCAG 705 A G A L A G Q R G I Y C F V T D G R G Q 2 3 5 CGTGTCJ^GGTGACTGCGGATGAGGCTTGTCGCATGCACAAGATCACCCCCGTCCAGTAT 765 R V K V T A D E A C R M H K I T P V Q Y 2 5 5 GAGCCCAAGGAGGCGCTTGGTCTG<;TCAACGGCACCGCTTTTTCAGCCTCTGTTGCGGGT 825 E P K E A L G L L N G T A F S A S V A G 2 7 5 CTCGCTACCTACGAGGCCGAAAATCTAGCCTCTCTGACGCAGCTCACCACCGCTATGGCC 885 L A T Y E A E N L A S L T Q L T T A M A 2 9 5 GTCGAAGCCCTCAAGGGTACCGATGCCAGCTTTGCTCCTTTCATTCACGAAATCGCCCGC 945 V E A L K G T D A S F A P F I H E I A R 3 1 5 CCGCATCCTGGTCAGATCAAGAGCGCCAAGTTTATCCGCGCGCATCTTTCCGGCTCTAGG 1005 P H P G Q I K S A K F I R A H L S G S R 3 3 5 CTAGCAGAGCATCTCGAAAACGAAAAGCACGTCCTCTTCTCCGAAGACAACGGAACGCTG 1 0 6 5 L A E H L E N E K H V L F S E D N G T L 3 5 5 C G T C A G G A C C G T T A C A C G C T G C A A A C C G C C T C C C A G T G G G T C G G C C C G G G T C T C G A G G A C 1125 R Q D R Y T L Q T A S Q W V G P G L E D 3 7 5 A T C G A A A A C G C A A A G C G A T C C G T C G A C T T T G A G A T T A A C A G C A C C A C A G A T A A C C C C A T G 1185 I E N A K R S V D F E I N S T T D N P M 3 9 5 A T C G A C C C G T A C G A C G G C G A C G G T C G C A T C C A C C A C G G A G G C A A C T T C C A G G C C A T G G C C 1245 I D P Y D G D G R I H H G G N F Q A M A 415 84 A T G A C G A A T G C C G T C G A G A A G A T C C G C C T C G C C T T G T G T G C T A T G G G C A A A A T G A C G T T C 1 3 0 5 M T N A V E K I R L A L C A M G K M T F 4 3 5 C A G C A G A T G I C A G A G C T C G T C A A C C C G G C A A T G A A C C G A G G A T T G C C C G C C A A C T T G G C T 1 3 6 5 Q Q M T E L V N P A M N R G L P A N L A 4 5 5 T C C A C G C C T G A T C T G T C G C T C A A C T T C C A C G C C A A G G G A A T C A A T A T T G C G C T T G C C A G T 1425 S T P D L S L N F H A K G I N I A L A S 4 7 5 G T C A C T T C G G A A C T C A T G T T C C T C G G C A A C C C C G T T T C A A C G C A T G T A C A A A G T G C A G A G 1485 V T S E L M F L G N P V S T H V Q S A E 4 9 5 A T G G C C A A C C A G G C C T T C A A C T C G C T G G C G C T C A T C A G C 1 G C C G C C A G A C G C T G C A G G C G 1545 M A N Q A F N S L A L I S G R Q T L Q A 5 1 5 A T C G A G T G C C T C T C G A T G A T T C A G G C T T G G T C G C T C T A C C T C T T G T G C C A A G C A C T C G A T 1 6 0 5 I E C L S M I Q A W S L Y L L C Q A L D 5 3 5 A T T C G C G C T T T G C A G T A T A A G G T T G C T G A G C A G C T G C C C A C G C T C A T C T T G G C A T C G C T G 1 6 6 5 I R A L Q Y K V A E Q L P T L I L A S L 5 5 5 C A C A G T C A C T T T G G C G A G T G G A T G G A T G A G A C C A A G C A G C A A G A G A T T G C A G C A C A G G T G 1725 H S H F G E W M D E T K Q Q E I A A Q V 5 7 5 C T C A A G A G C A T G A G C A A G C G T C T C G A C G A A A C C T C G T C C A A G G A C C T T C G C G A T C G A C T G 1785 L K S M S K R L D E T S S K D L R D R L 5 9 5 G T C G A G A C G T A C C A A G A C G C G T C G T C T G T G C T T G T G A G G T A C T T T T C C G A G C T G C C T A G C 1 8 4 5 V E T Y Q D A S S V L V R Y F S E L P S 615 G G T G G T G G T G C G G A T C C G C T G A G G A A C A T T G T C A A G T G G C G C G C C A C C G G T G T A G C T G A C 1 9 0 5 G G G A D P L R N I V K W R A T G V A D 635 A C G G A A A A G A T T T A C A G G C A G G T A A C G A T C G A A T T T C T T G A C A A C C C A T A C G C T T G C C A T 1 9 6 5 T E K I Y R Q V T I E F L D N P Y A C H 655 G C C A G C C A C C T G T T G G G C A A G A C C A A G C G C G C C T A C G A G T T T G T C A G G A A G A C G C T G G G T 2 0 2 5 A S H L L G K T K R A Y E F V R K T L G 675 G T G C C C A T G C A T G G T A A G G A G A A C C T C A A C G A A T T C A A G G G C G A A T T T G A G C A A T G G A A C 2 0 8 5 V P M H G K B N L N E F K G E F E Q W N 6 9 5 A C G A C G G G C G G T T A C G T C T C G G T C A T C T A T G C T A G T A T T C G A G A T G G C G A G T T G T A T A A C 2 1 4 5 T T G G Y V S V I Y A S I R D G E L Y N 7 1 5 A T G C T G A G C G A G C T C G A A A G G G A T T T G T A A A G G G G T G C A A G C A G C G T A T T A A T A G T T A G T 2 2 0 5 M L S E L E R D L 724 A T A A A T T G G C C A T C T A C G G T G A C A A A T T G C G T G T G A G T G C C A A A A G G G C C A T C G A A A T G A 2 2 6 5 T C A T G G A C A G C G A C A G A C T G T G T G T T G A T T T G T C A A A G T G A T T T G G C A C T A C C G A A T A T G 2 3 2 5 A C C G T G T G T A C C G G C A C C A A G G C G A G G T G A T G C G A A T G C A T G T T T T T G C G T G G C G T C A A A 2 3 8 5 G G G G G A T G C A G G A C A T G G T C G A C T G C T T G T C G G A G C T G A T G A G G T C G T A G C G G A T T C G G A 2 4 4 5 A T T T G G G T T C G A G G G C T G T G A A G G G A T G T T G A G G T G T A T C A A A G G G A C T T G G C T T G T G C T 2 5 0 5 G C G C T T G G G A G T G G G A G G G A C A T T T C A G G T G C A T C T G C T T T C G G G A T 2 5 5 2 Fig. 3.5. Complete nucleotide sequence of U. maydis PAL gene. Deduced amino acid sequence of the PAL protein is indicated below their respective codons. Numbers in the right margin indicate position relative to the A nucleotide of the start codon, or amino acid position. The start codon (ATG) and stop codon (TAA) are underlined. The putative TATA box in the 5' untranslated leader sequence is boxed. The PCR-amplified sequence is lettered in bold. 85 Table 3.3. BLAST search results with the deduced U. maydis PAL protein sequence. The results show the 30 most similar protein sequences in the GenBank for the deduced U. maydis PAL protein sequence. Rank Accession Organism Sequence Probability Number Description 1 P11544 Rhodosporidium toruloides PAL 2.7e-178 2 P10248 Rhodotorula rubra PAL 1.2e-172 3 P27991 Glycine max PAL 1 9.9e-94 4 P27990 Medicago sativa PAL 1.8e-90 5 P45726 Solanum tuberosum PAL 6.4e-90 6 P35510 Arabidopsis thaliana PAL 1 1.4e-89 7 P45732 Stylosanthes humilis PAL 1.5e-89 8 P31426 Camellia sinensis PAL 2 5.6e-89 9 P45730 Arabidopsis thaliana PAL 2.6e-88 10 P45728 Petroselinum crispum PAL 2 9.1e-88 11 P24481 Petroselinum crispum PAL 1 1.4e-87 12 P19142 Phaseolus vulgaris PAL Class II 2.5e-87 13 P25872 Nicotiana tabacum PAL 2.8e-87 14 P45729 Petroselinum crispum PAL 3 1.6e-86 15 Q01861 Pisum sativum PAL 1 4.3e-86 16 P45734 Trifolium subterraneum PAL 1.0e-85 17 P26600 Lycopersicon esculentum PAL 3.5e-84 18 P14717 Oryza sativa PAL 5.9e-84 19 P45733 Nicotiana tabacum PAL 7.0e-84 20 P45724 Arabidopsis thaliana PAL 2 1.7e-82 21 Q04593 Pisum sativum PAL 2 1.4e-82 22 P31425 Solanum tuberosum PAL 1 4.2e-82 23 P35511 Lycopersicon esculentum PAL 1.9e-81 24 P35513 Nicotiana tabacum PAL 3.9e-81 25 P45725 Arabidopsis thaliana PAL 3 6.5e-79 26 P45727 Persea americana PAL 5.6e-78 27 P45731 Populus kitakamiensis PAL 1.4e-77 28 P19143 Phaseolus vulgaris PAL Class III 4.7e-77 29 P14166 Ipomea batatas PAL 1.4e-75 30 P07218 Phaseolus vulgaris PAL Class I 3.4e-58 86 1 M A P T A D V L P P V E A S T R P G L L V Q P S D T K L R K A S S F R T E Q W I D G Y N L K I Q G L V A S A R Y G H V 61 T R P R P S A E T R K R I D D S V Q S L I A K L D G G E S I Y G I N T G F G G S A D S R T A N T R A L Q L A L L Q M Q Q 121 C G V L P V P S T F P T G E P S S A P F A L P L T D T E S S JL LIMOEAWVRG A I W R L A A L M RGHSGVRWEV 181 L D K M Q K L F L Q N N V T P W P V R S S I S A S G D L S P L S Y V A G A L A G Q R G I Y C F V T DGRGQRVKVT 2 4 1 A D E A C R M H K I T P V Q Y E P K E A * L G L L N G T A F S A S Y A G L A T Y E A E N L A S L T Q L T T A M A V E A L K 3 0 1 G T D A S F A P F I H E I A R P H P G Q L K S A K F I R A H L S G S R L A E H L E N E K H V L F S E DNGTLRQDRY 3 6 1 T L Q T A S Q W V G P G L E D I E N A K * R S V D F E I N S T TDNPMIDPYD GDGRIHHGGN FQAMAMTNAV 421 E K I R L A L C A M GKMTFQQMTE L V N P A M N R G L P A N L A S T P D L S L N F H A K G I N I A L A S V T S E L 4 8 1 M F L G N P V S T H V Q S A E M A N Q A F N S L A L I S G R Q T L Q A I E C L S M I Q A W S L Y L L C Q A L D I R A L Q 541 Y K V A E Q L P T L I L A S L H S H F G EWMDETKQQE I A A Q V L K S M S K R L D E T S S K D L R D R L V E T Y Q 601 D A S S V L V R Y F S E L P S G G G A D PLRNIVKWRA T G V A D T E K I Y R Q V T I E F L D N PYACHASHLL 6 6 1 G K T K R A Y E F V R K T L G V P M H G K E N L N E F K G E * FEQWNTTGGY V S V I Y A S I R D G E L Y N M L S E L 721 E R D L Fig. 3.6. Deduced amino acid sequence of U. maydis PAL. Amino acid sequence of an internal peptide fragment of the purified U. maydis PAL is underiined and the enzyme active site is indicated as U. Phenylalanine and histidine ammonia-lyases signature motif is boxed. Predicted N-glycosylation sites (* above line), cAMP- or cGMP- dependent protein kinase phosphorylation site (t), and ATP/GTP-binding site motif A (letters in bold) are shown. 87 are also predicted in the sequence (Fig. 3.6). The other potentially biologically significant sites are given in Table 3.4. Many putative phosphorylation sites are predicted. Codon usage of the U. maydis PAL gene is shown in Table 3.5. Codons that have a A in the wobble position are used infrequently, indicating that codon bias is operative in the U. maydis PAL gene, as in other fungal genes. Leucine, alanine, and serine appear as the three major amino acids in the deduced amino acid composition of U. maydis PAL (Table 3.6), while cysteine and tryptophan constitute less than 1 % of the gene. The hydropathic index of the PAL amino acid sequence in Fig. 3.7 shows that most of the amino acids are located in the positive end of the scale, suggesting the U. maydis PAL is a hydrophobic protein. 3.3.4 Genomic organization of PAL in U. maydis Southern blot hybridization using the 0.45 kb PAL fragment DNA as a probe was carried out to determine the number of PAL genes in the U. maydis genome. A single band was observed in each restriction digestion (Fig. 3.8). This suggests that U. maydis PAL is present as a single copy gene, as in the case of Rhodosporidium yeast PAL (Anson et al., 1987). 3.3.5 Northern blot analysis of PAL transcript Northern blot hybridization was performed by analyzing total RNA from the U. maydis cells harvested at the mid-log phase of growth. The 32P-labeled 0.45 kb PAL DNA probe hybridized to a 2.2 kb size of RNA (Fig. 3.9). This detected size is consistent with the 88 Table 3.4. Detection of potentially biologically significant sites in the protein sequence (724 aa) of U. maydis PAL. The putative sites were identified using PROSITE program from PC/GENE software system. Site Location : aa residues Tyrosine sulfation Protein kinase C phosphorylation Casein kinase II phosphorylation N-myristoylation 399 pmidp Y dgdgr 714 rdgel Y nmlse 14 ppvea S trpgl 69 dp sad T rkrid 323 pgqik S akf i r 354 sedng T lrqdr 508 s l a l i S grqtl 580 vlksm S krlde 587 rldet S skdlr 636 tgvad T ekiyr 663 hllgk T kraye 707 sviya S irdge 32 klrka S sseps 145 fal p l T dtess 240 qrvkv T adeac 389 dfein S ttdnp 456 panla S tpdls 587 rldet S skdlr 598 drive T yqdas 707 sviya S irdge 718 lynml S elerd 92 • gesiy G intgf 99 : ntgfg G sadsr 217 : lsyva G alagq 262 : pkeal G llngt 266 : l g l l n G taf sa 301 : vealk G tdasf 319 : arphp G qiksa 333 : rahls G srlae 409 : rihhg G nf qam 449 : pamnr G lpanl 468 : nfhak G i n i a l 484 : elmfl G npvst 632 : kwrat G vadte 698 : qwntt G gyvsv 89 Table 3.5. Codon usage in the U. maydis PAL gene CD AA No. PC CD AA Mo. PC CD AA No. PC CD AA No. PC TTT Phe 11 1 .5% TCT Ser 6 .8% TAT Tyr 4 .5% TGT Cys 3 .4% TTC Phe 12 1 . 6% TCC Ser 14 1 .9% TAC Tyr 15 2% TGC Cys 4 .5% TTA Leu 1 . 1% TCA Ser 4 .5% TAA 1 . 1% TGA 0 0% TTG Leu 12 1 .6% TCG Ser 17 2 .3% TAG 0 0% TGG Trp 7 .9% CTT Leu 12 1 .6% CCT Pro 8 1 .1% CAT His 7 .9% CGT Arg 11 1.5% CTC Leu 33 4 .5% CCC Pro 13 1 .7% CAC His 10 1 .3% CGC Arg 12 1.6% CTA Leu 2 .2% CCA Pro 7 .9% CAA Gin 6 .8% CGA Arg 8 1.1% CTG Leu 18 2 .4% CCG Pro 6 .8% CAG Gin 29 4% CGG Arg 0 0% ATT He 10 1 .3% ACT Thr 3 .4% AAT Asn 4 .5% AGT Ser 6 . 8% ATC He 23 3 . 1% ACC Thr 18 2 .4% AAC Asn 24 3 .3% AGC Ser 13 1.7% ATA He 0 0% ACA Thr 5 . 6% AAA Lys 3 .4% AGA Arg 0 0% ATG MET 21 2 .9% ACG Thr 20 2 .7% AAG Lys 27 3 .7% AGG Arg 10 1.3% GTT Val 6 .8% GCT Ala 17 2 .3% GAT Asp 13 1 .7% GGT Gly 24 3.3% GTC Val 23 3 .1% GCC Ala 29 4% GAC Asp 20 2 .7% GGC Gly 21 2.9% GTA Val 6 .8% GCA Ala 14 1 .9% GAA Glu 15 2% GGA Gly 4 .5% GTG Val 11 1 .5% GCG Ala 11 1 .5% GAG Glu 29 4% GGG Gly 2 .2% CD: codon, AA: amino acid, No.: number of given codons in the PAL gene, PC: percentage of given codons in the PAL gene, and --: stop codon. 90 Table 3.6. Inferred amino acid composition of U. maydis PAL Amino Acid No. % A l a 71 9.8 Arg 41 5.6 Asn 28 3.8 Asp 33 4.5 Cys 7 .9 Gin 35 4.8 Glu 44 6.0 G l y 51 7.0 His 17 2.3 H e 33 4.5 Leu 78 10.7 Lys 30 4.1 Met 21 2.9 Phe 23 3.1 Pro 34 4 . 6 Ser 60 8.2 Thr 46 6.3 Trp 7 .9 Tyr 19 2.6 V a l 46 6.3 No.: number of times an amino acid occurs. 91 Fig. 3.7. Hydropathy profile of U. maydis PAL from amino acid 1 to amino acid 724. Computation of the hydropathic index was performed with an interval of 5 amino acids, using the SOPE program in PC/GENE software system. A line at the -5 value divides hydrophobic regions above it from hydrophilic regions below. Arrow indicates the position of active site. 92 1 2 3 4 Fig. 3.8. Southern blot analysis of the U. maydis PAL gene. Genomic DNA from U. maydis strain 518 cells was digested with SamHI (lane 1), EcoRI (lane 2), Xoal (lane 3), and Xho\ (lane 4), fractionated by electrophoresis in a 0.7% agarose gel, transferred to Zeta-Probe membrane, and hybridized with 32P-labeled 0.45 kb PAL DNA fragment. Sizes of DNA markers are indicated in kb. 93 k b 9.4-7.4 — 4.4 — 2.4 -1.35 — 0.24 — PAL Fig. 3.9. Northern blot analysis of PAL transcript. Total RNA (20 uo,) was resolved in a 1% agarose-formaldehyde gel, blotted onto Zeta-Probe membrane, and hybridized with 32P-labeled 0.45 kb U. maydis PAL DNA fragment. Sizes of RNA markers are indicated in kb. 94 size of the open reading frame in the PAL DNA sequence in Fig. 3.5, and confirms that no introns are present in the U. maydis PAL gene. 3.3.6 Protein sequence comparison Protein sequences were obtained from the PIR and SWISS-PROT databases and used for the alignment and sequence identity comparison, using PC/GENE software. Four HAL protein sequences used were from Streptomyces griseus (a bacterium, database accession P24221), Pseudomonas putida (a bacterium, P21310), Rattus norvegicus (rat, P21213), and Homo sapiens (human, P42357). The ten PAL protein sequences were from Rhodosporidium toruloides (yeast, P11544), Rhodotorula rubra (yeast, P10248), Arabidopsis thaliana (P35510), Pinus taeda (pine, g1143312), Oryza sativa (rice, P14717), Triticum aestivum (wheat, g1483610), Bromheadia fmlaysoniana (orchid, g1491619), Solanum tuberosum (potato, P31426), Populus trichocarpa x Populus deltoides (poplar, P45730), and Medicago sativa (alfalfa, P27990). The percentage of protein sequence identity and similarity from a pairwise comparison of the above four HAL and eleven PAL protein sequences (including U. maydis PAL sequence) shows the extent of homology over various organisms (Table 3.7). As expected, sequences from closely- related organisms display the greatest protein sequence identity and similarity, e.g. between human and rat, or among potato, poplar and alfalfa. The lowest sequence conservation values (13-18%) are found between mammalian HALs and plant PALs. Overall, the U. maydis PAL shows low amino acid sequence identity with other PALs (23-26% with plant PALs, 39-40% with yeast PALs). It is quite noticeable that the level of homology (25%) shown between U. maydis PAL and bacterial HALs is similar to its 95 Table 3.7. Comparison of protein sequence identity and similarity of phenylalanine ammonia-lyase (PAL) and histidine ammonia-lyase (HAL) among different organisms. Numbers indicate the percentages of protein sequence identity/similarity. Sequences were analyzed based on inferred amino acid sequences from cDNAs encoding the two enzymes, using PALIGN program in the PC/GENE sequence analysis software, cn Plants I Fungi | Bacteria Mammals Alfalfa A. t. Potato Orchid Wheat Rice Pine U.m. R. t. R.r. S.g. P.p. Rat Human PAL PAL PAL PAL PAL PAL PAL PAL PAL PAL HAL HAL HAL HAL Poplar PAL 86/92 85/91 82/90 80/89 77/85 69/80 64/76 25/37 31/42 31/42 21/31 25/40 18/25 16/22 Alfalfa PAL 80/88 79/88 79/88 76/84 68/78 64/75 24/35 34/44 30/42 22/32 27/41 15/20 16/22 A. t. PAL 78/88 81/89 76/85 68/79 63/75 23/35 32/43 33/43 22/32 26/40 13/18 17/25 Potato PAL 78/88 76/85 68/79 63/75 24/35 31/41 28/38 19/30 24/39 15/21 15/21 Orchid PAL 77/86 69/80 64/76 26/38 30/42 27/38 19/30 26/39 14/20 15/21 Wheat PAL 70/81 65/76 25/36 33/45 31/42 22/31 24/37 13/18 13/19 Rice PAL 60/70 23/37 33/45 31/42 23/34 27/40 14/21 15/23 Pine PAL 25/38 28/39 28/39 22/32 22/34 14/20 13/17 C/.m. PAL 40/54 39/53 25/36 25/36 14/21 15/23 R. t PAL 77/87 25/36 25/38 20/27 18/25 R. r. PAL 25/36 26/39 17/26 17/26 S. g. HAL 38/53 37/52 38/52 P.p. HAL 41/55 42/55 Rat HAL 94/97 At:. Arabidopsis thaliana, U.m:. Ustilago maydis, R.t:. Rhodosporidium toruloides, R.r:. Rhodotorula rubra, S.t:. Streptomyces griseus, and, P. p.: Pseudomonasputida. identity with plant PALs (23-26%). This suggests a possible relatedness between U. maydis PAL and bacterial HAL. The protein sequence alignment results are in Fig. 3.10. The major conserved motifs are YGXXXGFG (X is any amino acid), ASGDLXPLS, PKEGLXXXNG, and EXNSXXDNP. Except for the ASGDLXPLS active site motif, the functional importance of these conserved motifs remains unknown. More variation among sequences is associated with in the amino- and carboxyl-terminal regions. The overall relation between HAL and PAL sequences over diverse organisms is displayed as a dendrogram in Fig. 3.11, which illustrates the divergence of these two ammonia-lyase enzymes from the putative ancestral form. 3.3.7 Immunological relationships To obtain further insights into the structural relationships between PAL and HAL proteins from different sources, Western blot analysis was used to determine the extent of cross-reactivity between these proteins from different species. Cross-reactivity was found among all the PAL proteins tested, using antibodies raised against both plant and fungal PALs (Fig. 3.12). Both samples of plant PAL antibodies strongly recognized plant PALs but only weakly recognized fungal PALs. Interestingly, however, antibodies raised against another fungal PAL (Ustilago), only weakly recognized the Rhodotorula glutinis (yeast) fungal PAL. Even more strikingly, although the anti-L/. maydis PAL antibodies showed low affinity for the plant PALs, they bound strongly to Pseudomonas bacterial 97 POPLARPAL ME TVTKNGYQNGSLES—LC 18 PALY_MEDSA ME TISAAITKNNANES—FCLIHA 22 PALY_ARATA ME INGAHKSNGGGVDA-MLCGGDI 23 PALl_SOLTU MA PSIAQNGHVNGEVEE-VL 19 ORCHIDPAL ME VSK EN-GLCL 11 WHEATPAL MA CAWRS 7 PALY_ORYSA MA GNGP 6 PINEPAL MV AAAEITQANEVQVKSTGLC 21 P AL Y_U S TMA MA PTADVLPPVEASTRPGLL VQ 22 PALY_RHOTO MA PSLDSISHS FANGVASAKQAVNG 25 PALY_RHORB MA PSVDSIATSVANSLSNGLHAAAA 25 HUTH_STRGR MDMHTVW 8 HUTH_PSEPU ME 2 HUTH_RAT MPRYTVHVRGEWLAVPCQDGKLSVGWLGREAVRRYMKNKPDNGGFTSVDE 50 HUMHISP MPRYTVHWGEWLAVPCQDAQLTVGWLGREAVRRYIKNKPDNGGFTSVDD 50 POPLARPAL PALY_MEDSA PALY_ARATA PALl_SOLTU ORCHIDPAL WHEATPAL PALY_ORYSA PINEPAL PALY_USTMA PALY_RHOTO PALY_RHORB HUTH_STRGR HUTH_PSEPU HUTH_RAT HUMHISP VNQRDPLSWGVAAE-KNNNNMKVNEADPLNWGVAAE-KTKN-MVINAEDPLNWGAAAE-WKKSIHDPLNWEMAVD-QGRDPLNWGAAAA-RSRA DPLNWGKAAE-INKEDPLNWGAAAA-TDFGSSGSDPLNWVRAAK-PSDTKLRK AST NLAVA GSHLP-ANGGDVHKKTAGA GSLLP-•AMKGSHLDEVKR-AMKGSHLDEVKR-QMKGSHLDEVKR-•SLRGSHLDEVKK-•ELQGSHLDEVKK-•ELSGSHLEAVKR-•EMAGSHLDEVKR-•AMEGSHFEEVKA-TTQVTQVDIVEK-TTETTQLDIVER-VRFLVRRCKGLGLLDNEDLLEVALEDNEFVEWIEGDVMSPDFIPSQPEG AHFLVRRCKGLGLLDNEDRLEVALENNEFVEWIEGDAMSPDFIPSQPEG 44 55 55 47 36 33 32 51 30 50 55 8 2 100 100 POPLARPAL —MVADYRKPV—VKLGGETLTIAQVASIAGHDT—GDVKVELSESA-RP 87 PALY_MEDSA —MVAEYRKPV—VRLGGET LTIS QVAAIAAHDH—G-VQVDLSESA-RD 97 PALY_ARATA —MVAEFRKPV—VNLGGET LT IGQVAAIS TIGN—S-VKVELSETA-RA 97 PALl_SOLTU —MVDEFRKPI—VKLWGETLTVAQVASIANADNKTSGFKVELSESA-RA 92 ORCHIDPAL —MVEEFRRPV—VKLEGVKLKISQVAAVAFGG GASAVELAESA-RA 78 WHEATPAL —MVEEYRKPV—VTMEGAT-TIAMVAAVAAG SDTRVELDESA-RG 73 P AL Y_0 RY S A —MVAQFREPL—VKIQGATLRVGQVAAVA-QAKDAARVAVELDEEA-RP 76 PINEPAL —MVDSYFGAKE-ISIEGKSLT-SDVAAVA RRSQVKVKLDAAAAKS 93 PALY_USTMA AS S FRTEQ-WT DGYNLKIQGLVASARYGHVTRPR PSAETRK 71 PALY_RHOTO —MLAAPTDST—LELDGYSLNLGDWSAARKGRPVRVK DSDEIRS 92 PALY_RHORB —ILADAGATDQ-1KLDGYTLTLGDWGAARRGRSVKVA DSPHIRE 98 HUTH_STRGR GTSGTTAEDWAVAR HGARVELSAAAVEA 37 HUTH_PSEPU LTLKPGTLTLAQLRAIHA APVRLQLDASAAPA 34 HUTH_RAT VFLYSKYREPEKYIALDGDSLSTEDLVNLGK GHYKIKLTSIAEKK 145 HUMHISP VYLYS KYREPEKYIELDGDRLTTEDLVNLGK GRYKIKLTPTAEKR 145 POPLARPAL PALY_MEDSA PALY_ARATA PALl_SOLTU ORCHIDPAL WHEATPAL PALY _ORYSA PINEPAL PALY USTMA GVKAS S DWVMD SMD KGT D S YGVTTGFGAT S H RR-GVKAS S EWYME SMNKGT D S YGVTTGFGAT S H S R-GVNAS S DWVME SMN KGT D S YGVTTGFGAT S H RR-GVKASSDWVMDSMSKGTDSYGVTTGFGATSHRR-GVKAS S DWVL E SVD KGT D S YGVTTGFGAT S H RR-RVKESSDWVMNSMMNGTDSYGVTTGFGATSHRR-RVKASSEWILTCIAHGGDIYGVTTGFGGTSHRR-RVEESSNWVLTQMTKGTDTYGVTTGFGATSHRR-RIDDSVQSLIAKLDGGESIYGINTGFGGSADSR-98 TKQGGALQKELIRFL 135 -TKQGGALQKELIRFL 145 TKNGVALQKELIRFL 145 TKNGGALQKELIKFL 140 -TKQGGALQKELIKFL 126 -TKEGGALQRELIRFL 121 TKDGPALQVELLRYL 124 TNQGAELQKELIRFL 141 TANTRALQLALLQMQ 119 PALY_RHOTO KIDKSVEFLRSQLS—MSVYGVTTGFGGSADTR—TEDAISLQKALLEHQ 138 PALY_RHORB KIDASVEFLRTQLD—NSVYGVTTGFGGSADTR—TEDAISLQKALLEHQ 144 HUTH_STRGR -LAAARLIVDALAAKPEPVYGVSTGFGALASRHIGTELRAQLQRNIVRSH 86 HUTH_PSEPU -IDASVACVEQIIAEDRTAYGINTGFGLLASTRIASHDLENLQRSLVLSH 83 HUTH_RAT -VQQSREVIDS11KERTWYGITTGFGKFARTVIPANKLQELQVNLVRSH 194 HUMHISP - VQKS REVI DS 11KEKTWYGI TTGFGKFARTVI PINKLQELQVNLVRSH 194 POPLARPAL NAGIFGNG TETCHTLPHSATRAAMLVRINT 165 PALY_MEDSA NAGIFGNG TESNHTLP KTAT RAAMLVRI NT 175 PALY_ARATA NAGIFGST KETSHTLPHSATRAAMLVRINT 75 PALl_SOLTU NAGVFGNG TESTHTLPHSATRAAMLVRINT 17 0 ORCHIDPAL NAGIFGSG N—SNTLPSAATRAAMLVRINT 154 WHEATPAL NAGAFGTG TDG-HVLPAAATRAAMLVRVNT 150 PALY_ORYSA NAGIFGTG SDG-HTLPSETVRAAMLVRINT 153 PINEPAL NAGVLGKC PE—NVLSEDTT-AAMLVRTNT 168 PALY_USTMA QCGVLPVP-STFPTGEPSSAPFALPLTDTESSLIMPEAWVRGAIWRLSS 168 PALY_RHOTO LCGVLPSSFDSFRLGRGLENSLPLEV VRGAMTIRVNS 175 PALY_RHORB LCGVLPTSMDGFALGRGLENSLPLEV VRGAMTIRVNS 181 HUTH_STRGR AAGM GPRVEREWRALMFLRLKT 109 HUTH_PSEPU AAGI GAPLDDDLVRLIMVLKINS 106 HUTH_RAT SSGV GKPLSPERCRMLLALRINV 217 HUMHISP SSGV GKPLSPERCRMLLALRINV 217 POPLARPAL LLQGYSGIRFEILEAITRL-LNNNITPCLPLRGTITASGDLVPLSYIAGL 214 PALY_ME D SA LLQGYSGIDFEILEAITKP-LNKTVTPCLPLRGTITASGDLVPLSYIAGL 224 PALY_ARATA LLQGFSGIRFEILEAITSF-LNNNITPSLPLRGTITASGDLVPLSYIAGL 224 PALl_SOLTU LLQGYSGIRFEILEAITKL-INSNITPCLPLRGTVTASGDLVPLSYIAGL 219 ORCHIDPAL LLQGYSGIRFEILKAIATL-LNKNITPCLPLRGTITASGDLVPLSYLAGI 203 WHEATPAL LLQGYSGIRFEILETIATL-LNANVTPCLPLRGTITASGDLVPLSYIAGL 199 PALY_ORYSA LLQGYSGIRFEILEAITKL-LNTGVTPCLPLRGTITASGDLVPLSYIAGL 202 PINEPAL LLQGYSGVRWDILETVEKL-LNAWLTPKLPLRGTITASGDLVPLSYIAGL 217 PALY_USTMA LMRGHSGVRWEVLDKMQKLFLQNNVTPWPVRSSISASGDLSPLSYVAGA 218 PALY_RHOTO LTRGHSAVRLWLEALTN-FLNHGITPIVPLRGTISASGDLSPLSYIAAA 224 PALY_RHORB LTRGHSAVRIWLEALTN-FLNHGIT PIVPLRGTISASGDLSPLSYIAAS 230 HUTH_STRGR VASGHTGVRPEVAQTMADVL-NAGITPWHEYGSLGCSGDLAPLSHCALT 158 HUTH_PSEPU LSRGFSGIRRKVIDALIALV-NAEVYPHIPLKGSVGASGDLAPLATMSLV 155 HUTH_RAT LAKGYSGISLETLKQVIEVF-NASCLSYVPEKGTVGASGDLAPLSHLALG 266 HUMHISP LAKGYSGISLETLKQVIEMF-NASCLPYVPEKGTVGASGDLAPLSHLALG 266 _ * _ _ _ _ ** + + *+_ _ POPLARPAL PALY_MEDSA PALY_ARATA PALl_SOLTU ORCHIDPAL WHEATPAL PALY_ORYSA PINEPAL P AL Y_U S TMA PALY_RHOTO PALY_RHORB HUTH STRGR -VLDAAEAFKAAGIESGFFELQPKEGLALVNG 260 -VLNAKEAFNLAGINAEFFELQPKEGLALVNG 270 -ALTAEEAFKLAGISSGFFDLQPKEGLALVNG 270 -KLDADEAFRVAAVSGGFFELQPKEGLALVNG 265 -TVDATTAFRLAGIS S GFFDLQPKEGLALVNG 249 -KVNAAEAFKIAGIQHGFFELQPKEGLAMVNG 245 -KVDAAEAFKLAGIEGGFFTLNPKEGLAIVNG 248 -EMSGAEALKKVGLEKPF-ELQPKEGLAIVNG 262 LAGQRGIYCFVTDGRGQRVKVTADEACRMHKITP—VQYEPKEALGLLNG 266 ISGHPDS KVHWHEGKEKI - LYAREAMALFNLEP—WLGPKEGLGLVNG 271 ITGHPDSKVHV DGKI -MSAQEAIALKGLQP—WLGPKEGLGLVNG 273 LMG- - EGEAEGPDGT VRPAGELLAAHGIAP—VELREKEGLALLNG 200 LTGRPNSKATGPTGE-LTGRPNSKAHGPSGE-LTGRPNSKATGPNGE-LTGRPNSKAVGPSGS LTGRPNSKARTPNGS VTGRPNSMATAPDGS ITGRPNAQAISPDGR LT GRPN S RVRS RDGI • 99 HUTH_PSEPU HUTH_RAT HUMHISP LLG—EGKARY-KGQ-LIG—EGKMWSPKSG-LVG—EGKMWSPKSG--WLSATEALAVAGLEP—LTLAAKEGLALLNG 196 -WADAKYVLEAHGLKP—IVLKPKEGLALING 308 -WADAKYVLEAHGLKP—VILKPKEGLALING 308 _ _ * + _* ** POPLARPAL TAVGSGLASMVLFETNVLAVLSELLSAIFAEVMNGKPE-FTDHLTHKLKH 309 PALY_MED SA TAVGSGLASIVLFEANILAVLSEVLSAIFAEVMQGKPE-FTDHLTHKLKH 319 PALY_ARATA TAVGSGMASMVLFETNVLSVLAEILSAVFAEVMSGKPE-FTDHLTHRLKH 319 PAL1_S0LTU TAVGSGMASIVLYDSNILAVMFEVLSAIFAEVMNGKPE-FTDYLTHKLKH 314 ORCHIDPAL TAVGSGVASIVLFETNILAVMAELLSALFCEVMQGKPE-FTDHLTHKLKH 298 WHEATPAL TAVGSGLASMVLFEANVLSLLAEVLSGVFCEVMNGKPE-FTDHLTHKLKH 294 PALY_ORYSA TSVGSALAATVMFDANILAVLSEVLSAVFCEVMNGKPE-YTDHLTHKLKH 297 PINEPAL TSVGAALASIVCFDANVLALLSEVISAMFCEVMNGKPE-FTDPLTHKLKH 311 PAL Y_U S TMA TAFSASVAGLATYEAENLASLTQLTTAMAVEALKGTDASFAPFIHEIARP 316 PALY_RHOTO TAVS ASMAT LALH DAHML S L L S Q S LT AMT VEAMVGHAG S FH P FLH DVT RP 321 P AL Y_RHO RB TAVS ASMAT LALT DAHVL S L LAQALT ALT VEAMVGHAG S FH P FLH DVT RP 323 HUTH_STRGR TDGMLGMLVMALADLRNLYTSADITAALSLEALLGTDKVLAPEL-HAIRP 249 HUTH_PSEPU TQASTAYALRGLFYAEDLYAAAIACGGLSVEAVLGSRSPFDARI-HEARG 245 HUTH_RAT TQMITSLGCEAVERASAIARQADIVAALTLEVLKGTTKAFDTDI-HAVRP 357 HUMHISP TQMITSLGCEAVERASAIARQADIVAALTLEVLKGTTKAFDTDI-HALRP 357 POPLARPAL PALY_MEDSA PALY_ARATA PAL1_S0LTU ORCHIDPAL WHEATPAL PALYJDRYSA PINEPAL PALY_USTMA PALY_RHOTO PALY_RHORB HUTH_STRGR HUTH_PSEPU HUTH_RAT HUMHISP HPGQ-IEAAAIMEHILDGSAYMKAAKKLHETDPLQKP-HPGQ-1EAAAIMEHILDGS SYVKAAKKLHEIDPLQKP-HPGQ-IEAAAVMEHILDGS SYMKLAQKLHEMDPLQKP-HPGQ-IEAAAIMEHILDGSSYVKAAQKLHEMDPLQKP-HPGQ-1EAAAVMEHILEGS SYMKMAKKLHEMDPLQKP-HPGQ-IEAAAIMEHILEGS SYMMLAKKLGELDPLMKP--KQDRYALR -KQDRYALR -KQDRYALR -KQDRYALR -KQDRYALR -KQDRYALR HPGS-IDAAAIMEHILAGSSFMSHAKKVNEMDPLLKP KQDRYALR HPGQ-MEAAAIMEYVLDGSSYMKHAAKLHEMNPLQKP KQDRYGLR HPGQ-IKSAKFIRAHLSGSRLAEHLE—NEKHVLFSEDNGTLRQDRYTLQ HPTQ-IEVAGNIRKLLEGSRFAVHHE—EEVKV—KDDEGILRQDRYPLR HPTQ-IEVARNIRTLLEGSKYAVHHE—TEVKV—KDDEGILRQDRYPLR HPGQGVSADNMSRVLAGSG LTGHHQDDAPR VQDAYSVR QRGQ-IDTAACFRDLLG DSSEVSLSHKNCDKVQDPYSLR HRGQ-IEVAFRFRSLLDSD HHPSEIAESHRFCDRVQDAYTLR HRGQ-1EVAFRFRS LLDS D HHPSEIAESHRFCDRVQDAYTLR _ _ _ _ ** * 353 363 363 358 342 338 341 355 363 366 368 287 283 398 398 POPLARPAL TSPQWLGPQIEVIRFSTKSIEREI-NSVNDNPLIDV—SRNKAIHGGNFQ 400 PALY_MEDSA TSPQWLGPLVEVIRFSTKSIEREI-NSVNDNPLIDV—SRNKALHGGNFQ 410 PALY_ARATA TS PQWLGPQIEVIRYATKSIEREI-NSVNDNPLIDV—SRNKAIHGGNFQ 410 PALl_SOLTU TSPQWLGPQIEVIRAATKMIEREI-NSVNDNPLIDV—SRNKAIHGGNFQ 405 ORCHIDPAL T S PQWLGPQIEVIRAATKSIEREI-NSVNDNPLIDV—SRNKALHGGNFQ 389 WHEATPAL TSPQWLGPQIEVIRAATKSIEREI-NSVNDNPLIDV—SRGKAIHGGNFQ 385 PALY_ORYSA TSPQWLGPQIQVIRAATKSIEREV-NSVNDNPVIDV—HRGKALHGGNFQ 388 PINEPAL TS PQWLGPQVEIIRSATHMIEREI-NSVNDNPVIDV—ARDKALHGGNFQ 402 PALY_USTMA TASQWVGPGLEDIENAKRSVDFEI-NSTTDNPMIDPYDGDGRIHHGGNFQ 412 PALY_RHOTO TSPQWLGPLVSDLIHAHAVLTIEAGQSTTDNPLIDV—ENKTSHHGGNFQ 414 PALY_RHORB CSPQWLGPLVSDMIHAHAVLSLEAGQSTTDNPLIDL—ENKMTHHGGAFM 416 HUTH_STRGR CAPQVNGAGRDTLDHAALVAGREL-ASSVDNPW—LPD-GRVESNGNFH 333 HUTH_PSEPU CQPQVMGACLTQLRQAAEVLGIEA-NAVSDNPLV—FAAEGDVISGGNFH 330 HUTH_RAT CCPQVHGWNDTIAFVKDIITTEL-NSATDNPMV— FAS RGETI SGGNFH 445 HUMHISP CCPQVHGWNDTIAFVKNI ITTEL-NSATDNPMV—FANRGETVSGGNFH 445 100 POPLARPAL GTPIGVSMDNVRLAIASIGKLLFAQFSELVNDFYNNGLPSNLTASRNPSL 450 PALY_MEDSA GTPIGVSMDNTRLALASIGKLMFAQFSELVNDFYNNGLPSNLSASRNPSL 460 PALY_ARATA GTPIGVSMDNTRLAIRAIGKLMFAQFSELVNDFYNNGLPSNLTASRNPSL 460 PALl_SOLTU GTPIGVSMDNTRLALASIGKLMFAQFSELVNDYYNNGLPSNLTAGRNPSL 455 ORCHIDPAL GTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSSGRNPSL 439 WHEATPAL GTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSL 435 PALY_ORYSA GTPIGVSMDNARLAIANIGKLMFAQFSELVNEFYNNGLTSNLAGSRNPSL 438 PINEPAL GTPIGVSMDNLRLSISAIGKLMFAQFSELVNDYYNGGLPSNLSGGPNPSL 452 PALY_U S TMA AMAMTNAVEKIRLALCAMGKMTFQQMTELVNPAMNRGLPANLASTPDLSL 462 PALY_RHOTO AAAVANTMEKTRLGLAQIGKLNFTQLTEMLNAGMNRGLPSCLAAE-DPSL 463 PALY_RHORB ASSVGNTMEKTRLAVALMGKVSFTQLTEMLNAGMNRALPSCLAAE-DPSL 465 HUTH_STRGR GAPVAYVLDFLAIVAADLGSICERRTDRLLDKNRSHGLPPFLAD—DAGV 381 HUTH_PSEPU AEPVAMAADNLALAIAEIGSLSERRISLMMDKHMSQ-LPPFLVE—NGGV 377 HUTH_RAT GEYPAKALDYLAIGVHELAAISERRIERLCNPSLSE-LPAFLVA—EGGL 492 HUMHISP GEYPAKALDYLAIGIHELAAISERRIERLCNPSLSE-LPAFLVA—EGGL 492 POPLARPAL DYGFKGAEIAMASYCSELQYLANPVTTHVQSAEQHNQDVNSLGLISSRKT 500 PALY_MED S A DYGFKGAEIAMASYCSELQYLANPVTTHVQSAEQHNQDVNSLGLISARKT 510 PAL Y_ARATA DYGFKGAEIAMASYCSELQYLANPVTSHVQSAEQHNQDVNSLGLISSRKT 510 PALl_SOLTU DYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISARKT 505 ORCHIDPAL DYGFKGAEIAMASYCSELQALANPVTNHVQSAEQHNQDVNSLGLISSRKT 489 WHEATPAL DYGFKGAEIAMASYCSELQFLGNPVTNHVQSAEQHNQDVNSLGLISSRKT 485 PALY_ORYSA DYGFKGTEIAMASYSSELQYLANPITNHVQSAEQHNQDVNSLGLVSARKT 488 PINEPAL DYGLKGAEIAMASYTSELLYLANPVTSHVQSAEQHNQDVNSLGLVSARKS 502 PALY_USTMA NFHAKGINIALASVTSELMFLGNPVSTHVQSAEMANQAFNSLALISGRQT 512 PALY_RHOTO SYHCKGLDIAAAAYTSELGHLANPVTTHVQPAEMANQAVNSLALISARRT 513 PALY_RHO RB S YH C KGLDIAAAAYT S ELGHLAN PVS THVQPAEMGNQAINSLALISARRT 515 HUTH_STRGR DSGLMIAQYTQAALVSEMKRLAVPASADSIPSSAMQEDHVSMGWSAARKL 431 HUTH_PSEPU NSGFMIAQVTAAALASENKALSHPHSVDSLPTSANQEDHVSMAPAAGKRL 427 HUTH_RAT NSGFMIAHCTAAALVSESKALCHPSSVDSLSTSAATEDHVSMGGWAARKA 542 HUMHISP NSGFMIAHCTAAALVSENKALCHPSSVDSLSTSAATEDHVSMGGWAARKA 542 _ _ _ _ *_ ** * * _ _ * POPLARPAL AEAVDILKLMSTTFLVALCQAIDLRHLEENLKSAVKNTVSQVSKRVLTTG 550 PALY_MEDSA NEAIEILQLMSSTFLIALCQAIDLRHLEENLKNSVKNTVSQVAKKTLTMG 560 PALY_ARATA SEAVDILKIiMSTTFLVAICQAVDLRHLEENLRQTVKNTVSQVAKKVLTTG 560 PALl_SOLTU AEAVDILKLMS STYLVALCQAIDLRHLEENLKSWKNTVSQVAKRTLTIG 555 ORCHIDPAL AEAVDI LKLMSTTFLVGLCQAVDLRHLEENLKNAVKNTVSQVAKRVLTMG 539 WHEATPAL AEAIDILKLMSSTFLVALCQAIDLRHLEENVKNAVKSCVKTVARKTLSTD 535 PALY_ORYSA LEAVDILKLMTSTYIVALCQAVDLRHLEENIKSSVKNCVTQVAKKVLTMN 538 PINEPAL AEAIDILKLMLSTYLTALCQAVDLRHLEENMLATVKQIVSQVAKKTLSTG 552 PALY_U S TMA LQAIECLSMIQAWSLYLLCQALDIRALQYKVAEQLPTLILASLHSHFGEW 562 PALY_RHOTO TESNDVLSLLLATHLYCVLQAIDLRAIEFEFKKQFGPAIVSLIDQHFGSA 563 PALY_RHORB AEANDVLSLLLATHLYCVLQAVDLRAMEFEHTKAFEPMVTELLKQHFGAL 565 HUTH_STRGR RTAVDNLARIVAVELYAATRAIELRAAE-GLTPAPA SEAWAALRAA 477 HUTH_PSEPU WEMAENTRGVPAIEWLGACQGLDLR—K-GLKTSAK LEKARQALRSE 471 HUTH_RAT LRVIEHVEQVLAIELLAACQGIEFL—R-PLKTTTP LEKVYDLVRSV 586 HUMHISP LRVIEHVEQVLAIELLAACQGIEFL—R-PLKTTTP LEKVYDLVRSV 586 POPLARPAL ANGELHPSRFCE KELLKWDREYVFAYVDDPCSATYPLMQKLRQVFV 597 P AL Y_ME D S A VNGELHPSRFCE KDLLKWDREHVFAYIDDPCSATYPLSQKLRQVLV 607 PALY_ARATA VNGELHPSRFCE KDLLKWDREQVYTYADDPCSATYPLIQKLRQVIV 607 PALl_SOLTU AIGELHPARFCE KELLRWDREYLFTYADDPCSSTYPLMQKLRQVLV 602 ORCHIDPAL VNGELHPSRFCE KDLIKVIDREYVFAYADDPCSSTYPLMQKLRAVIV 586 WHEATPAL NNGHLHNARFCE KDLLLTIDREAVFAYADDPCSANYPLMQKMRAVLV 582 101 PALY_ORYSA PTGDLSSARFSE KNLLTAIDREAVFSYADDPCSANYPLMQKLRAVLV 585 PINEPAL LNGELLPGRFCE KDLLQWDNEHVFSYIDDPCNASYPLTQKLRNILV 599 PALY_U S TMA MDETK-QQEIAAQVLKSMSKRLDETSSKDLRDRLVETYQDASSVLVRYFS 611 PALY_RHOTO MTGSNLRDELVEKVNKTLAKRLEQTNSYDLVPRWHDAFSFAAGTWEVLS 613 PALY_RHORB ATA EVEDKVRKSIYKRLQQNNSYDLEQRWHDTFSVATGAWEALA 610 HUTH_STRGR GAEGPGPDRFLAPDLAAADTFVREGRLVAAVE PVTG 513 HUTH_PSEPU -VAHYDRDRFFAPDIEKAVELLAKGSLTGL 500 HUTH_RAT -VRPWIKDRFMAPDIEAAHRLLLDQKVWEVAA PYIEKYR 624 HUMHISP -VRPWIKDRFMAPDIEAAHRLLLEQKVWEVAA PYIEKYR 624 POPLARPAL DHALENGENEKNFSTSVFQKIEAFEEELKALLPKEVESARAAYDSGNSAI 647 PALY_MEDSA DHALVNGESEKNFNTSIFQKIATFEEELKTLLPKEVESARTAYESGNPTI 657 PALY_ARATA DHALVNGESEKNAVTSIFHKIGAFEEELKAVLPKEVEAARAAYDNGTSAI 657 PALl_SOLTU DHAMKNGESEKNINSSIFCjKIGAFEDELNAVLPKEVESARALLESGNPSI 652 ORCHIDPAL EHALNNGVKEKDSNTSIFQKIS S FENELKAALPKEVEAARAEFENGS PAI 636 WHEATPAL EHALANGE-EAHVETSVFAKIJWFEQELRAVLPKEVEAARSAVENGTAAQ 631 PALY_0 RY SA EHALTSGDR-RARGLRVLQDHQVRGGAPLCAAPGDRGRPRRRRQR-TAPV 633 PINEPAL EHAFKNAEGEKDPNTSIFNKIPVFEAELKAQLEPQVS LARESYDKGT S PL 649 PALY_U S TMA E—LPSGGGADPLRNIVKWRATGYADTEK—IYRQVTIEFLDNPYACHAS 657 PALY_RHOTO STSLSLAAVNAWKVAAAESAIS—LTRQVRETFWSAASTSSPA 654 PALY_RHORB GQEVS LAS LNAWKVAGAEKAIA—LTRSVRDSFWAAPSSSSPA 651 HUTH_STRGR 513 HUTH_PSEPU 500 HUTH_RAT 624 HUMHISP 624 POPLARPAL PALY_MED SA PALY_ARATA PAL1_S0LTU ORCHIDPAL WHEATPAL PALYJDRYSA PINEPAL PALYJJSTMA PALY_RHOT O PAL Y_RHO RB HUTH_STRGR HUTH_PSEPU HUTH_RAT HUMHISP DNKIKECRSYPLYKFVREELGTVLLTGEKVQS PGEEFDKVFTAMCQGKII PNKINGCRSYPLYKFVREELGTGLLTGENVISPGEECDKLFSAMCQGKII PNRIKECRSYPLYRFVREELGTELLTGEKVTSPGEEFDKVFTAICEGKII PNRITECRSYPLYRLVRQELGTELLTGEKVRSPGEEIEKVFTAMCNGQIN ENRIKDCRSYPLYKFVKE-VGSGFLTGEKWSPGEEFDKVFNAICEGKAI QNRIAECRSYPLYRFVRKELGTEYLTGEKTRSPGEEVDKVFVAMNQGKHI ANRIVES RS FPLYRFVREELGCVFLTGEKLKS PGEECNKVFLGISQGKLI PDRIQECRS YPLYEFVRNQLGTKLLSGTRTIS PGEVIEWYDAISEDKVI —HLLG-KTKRAYEFVRKTLGV PMHGKEN L LSYLSP-RTQILYAFVREELGV KAR-RGD V LKYLS P- RTRVLYS FVREEVGV KAR-RGD V p LA LPAGVLP —LSPTAFSLESLRKNSATIPESD —LSPTAFSLQFLHKKSTKIPESE MEHIPESRP-MEHIPESRP-697 707 707 702 685 681 683 699 684 682 679 516 507 655 655 POPLARPAL PALY_MEDSA PALY_ARATA PALl_SOLTU ORCHIDPAL WHEATPAL PALY_ORYSA PINEPAL PALY_USTMA PALY_RHOTO PALY_RHORB HUTH_STRGR HUTH_RAT HUMHISP DPMLECLGEWNGS PLPIC DPLLECLGEWNGA PLPIC DPMMECLNEWNGA PIPIC • DPLLECLKSWNGA PLPIC DPMLDCLKEWNGA PLPIC DALLECLKEWNGE PLPLC DPMLDCLKEWNGE PLPIN VPLFKCLDGWKGTLAHSEINNLPRSPLYNDCYDLSPRMLLLMLLFSDPEF NEFKGEFEQWNTTGGY VSVIYA SIRDGELYNMLSELER —FLGKQEV—TIGSN VSKIYE AIKSGRINNVL—LKM —YLGKQEV—TIGTN VSRIYE AIKSGCIAPVL—VKM S L D L D L 715 725 725 720 703 699 701 749 722 714 711 516 509 657 102 POPLARPAL PALY_MEDSA PALY_ARATA PALl_SOLTU ORCHIDPAL WHEATPAL PALY_ORYSA PINEPAL P AL Y_U S TMA PALY_RHOTO PALY_RHORB HUTH_STRGR HUTH_PSEPU HUTH_RAT HUMHISP 715 725 725 720 703 699 701 DWS 752 D-L 724 L-A 716 M-A 713 516 509 657 657 Fig. 3.10. Comparison of the deduced amino acid sequence of U. maydis PAL with the deduced amino acid sequences of PAL and HAL from various organisms. PALs; Poplar (POPLARPAL), Alfalfa (PALY_MEDSA), A. thaliana (PALY_ARATA), Potato (PAL1_SOLTU), Orchid (ORCHIDPAL), Wheat (WHEATPAL), Rice (PALYJ0RYSA), Pine (PINEPAL), U. maydis (PALY_USTMA), R. toruloides (PALY_RHOTO), and R. rubra (PALY_RHORB). HALs; S. griseus (HUTH_STRGR), P. putida (HUTH_PSEPU), Rat (HUTH_RAT), and Human (HUMHISP). The amino acid sequences are in one-letter code and have been aligned using the Clustal program in PC/GENE software. The protein sequences were obtained from the SWISS-PROT database. Their database accession numbers are described in Materials and Methods. Perfectly conserved and well- conserved positions in the alignment are indicated as * and -, respectively. Bold sequences indicate highly conserved regions. 103 Organisms Dicot Angiosperm PAL Plants Mnnncnt Mnnomt Mnnncnt Gymnosperm Fungi Filamentous Poplar Alfalfa Arabidopsis Potato Orchid Wheat Rice Pine Ustilago maydis Rhodosporidium toruloides Rhodotorula rubra Streptomyces griseus Pseudomonas putida Rat Human Fig. 3.11. Dendrogram of the inferred protein sequences of PALs and HALs from various organisms using PC-GENE Clustal Program. PALs; Poplar (POPLARPAL), Alfalfa (PALY_MEDSA), Arabidopsis thaliana (PALY_ARATA), Potato (PAL1_SOLTU), Orchid (ORCHIDPAL), Wheat (WHEATPAL), Rice (PALYJORYSA), Pine (PINEPAL). U. maydis (PALY_USTMA), R. toruloides (PALY_RHOTO), and R. rubra (PALY_RHORB). HALs; S. griseus (HUTH_STRGR), P. putida (HUTH_PSEPU), Rat (HUTH_RAT), and Human (HUMHISP). Yeasts HAL Oram Positive Racteria Gram Negative Rarteria Mammals Fig. 3.12. Native PAGE (top) and Western blots (center and bottom) of fungal PAL, plant PAL and bacterial HAL using polyclonal antibodies raised against U. maydis PAL (A), poplar PAL (B), alfalfa PAL (C), P. fluorescens HAL (D), and P. putida HAL (E). Proteins loaded: Lane1-l/. maydis PAL, Lane 2-R. toruloides PAL, Lane 3-poplar PAL, Lane 4-potato PAL, and Lane 5-P. fluorescens HAL. Arrow indicates the location of PAL or HAL protein. 105 106 HAL. Significant cross-reactivity between two plant PAL antibodies and the bacterial HAL was also observed. Immunological enzyme inhibition tests were conducted to see whether the pattern of cross-reactivity observed on western blots carried over to solution reactions (Table 3.8). In this system, both the ani\-Ustilago PAL and the anti-poplar PAL antibodies displayed similar enzyme inhibition patterns, including moderate inhibition of bacterial HAL activity. However, the bacterial HAL antibody inhibited only Ustilago PAL. The PAL and HAL antibodies tested showed no inhibition against yeast PAL. The hydrophilicity profiles of the entire deduced amino acid sequences of poplar PAL, alfalfa PAL, Rhodotorula yeast PAL, Ustilago PAL, Pseudomonas bacteria HAL and rat HAL were analyzed using PC/GENE software program to compare the location of putative antigenic determinant sites (Fig. 3.13). Ustilago PAL contains two sites with high hydrophilicity but the other sequences contain only one site, suggesting that at least some antigenic determinant sites of Ustilago PAL would be different from those of the other enzymes compared. An overall pattern of similarity is present only between the two plant PALs and yeast PAL in that the most hydrophilic point is located in the middle of the sequences in each case. By contrast, the most hydrophilic sites of U. maydis PAL are located closer to the amino- and carboxyl-terminal ends. It is noticeable that the most hydrophilic site of bacterial HAL is located closer to the carboxyl-terminal of the sequence, as in Ustilago PAL. 107 Table 3.8. Inhibition of PAL and HAL activity by antisera raised against the two ammonia-lyases Enzymes P. fluorescens HAL antiserum U. maydis PAL antiserum Poplar PAL antiserum P. fluorescens HAL U. maydis PAL R. toruloides PAL Potato PAL Poplar PAL + (68%) + (90%) + (84%) + (60%) + (39%) + (74%) + (80%) + (92%) + (67%) + (80%) +: Inhibition, -: no inhibition, (%): inhibition of enzyme activity by antiserum expressed as a percentage of a control activity with preimmune serum. 108 Fig. 3.13. Hydrophilicity profiles of PALs and HALs from different organisms. A: alfalfa PAL, B: poplar PAL, C: R. toruloides PAL, D: U. maydis PAL, E: P. putida HAL, and rat HAL. Positive values indicate hydrophilic regions. Vertically dotted lines indicate the most hydrophilic amino acid residues. Arrows indicate the position of acive site. 109 3.4 DISCUSSION PAL genes have been cloned from numerous cultivated plant species as well as two trees and a weed but only from two red yeasts. Today many potential methods are available to clone PAL genes from plants and yeast. So far, most PAL genes described in the literature have been cloned by one of three approaches: 1) using antibodies raised against the purified enzyme from the same species, 2) using heterologous DNA probes from other species, or 3) using PCR amplification with degenerate oligonucleotide primers based on amino-terminal peptide sequences of purified PAL or conserved amino acid sequences among different species. Initial trials with these approaches for the isolation of a PAL cDNA clone from U. maydis cDNA libraries were unsuccessful. Immunoscreening of libraries with anti-L/. maydis PAL antibodies produced non-specific background, while no hybridization signal was detected with poplar and yeast PAL DNA probes. Purified U. maydis PAL enzyme was found to be blocked at the amino terminus, preventing protein sequencing. Internal peptide sequences obtained by tryptic digestion were found to have high codon degeneracy and primers based on these sequences had low calculated annealing temperatures, so cloning based on this information was also unsuccessful. Based on the known primary structure of PAL genes from plants and yeast, several degenerate oligonucleotide primers were designed, and two of these succeeded in amplifying a U. maydis PAL DNA fragment (0.45 kb) by PCR from a U. maydis genomic DNA template. Subsequently, a PAL gene was isolated by using this 0.45 kb fragment as a probe to screen a U. maydis genomic library. As shown in Fig. 3.2, this PCR approach n o was successful only with the combination of the enzyme active-site primer, UMPAL7, and a yeast sequence-specific primer, RTPAL3. It is worth noting that combinations of oligonucleotide primers based on other highly conserved sequences among PAL genes from various species failed to amplify U. maydis PAL in PCR. PAL is known to be a difficult enzyme to purify sufficiently for the production of antibodies (Jones, 1984), and even then, the antibody screening methods are not always successful (as in the case of this study). The failing of most of the degenerate primer combinations shows how challenging the cloning of highly diverged PAL genes can be. The 2172 bp nucleotide genomic DNA sequence determined in this study is confirmed to be a PAL gene on the basis of its homology with other known PALs, conservation of enzyme active site sequences, identity with the internal peptide sequence of purified PAL protein, and similarity of physico-chemical properties (e.g. molecular weight and isoelectric point) predicted from the deduced protein sequence with those of the purified enzyme. Among PAL enzymes purified to date, the isoelectric point of 6.3 found for purified U. maydis PAL (chapter 2) makes this the least acidic of the known PAL proteins. Interestingly, this value corresponds to the calculated value of 6.3 from the deduced protein sequence. Protein data from many sources show that PAL is a homo-tetrameric protein consisting of four identical subunits with molecular weights in the range of 72000-83000 (Schomburg and Salzmann, 1990). In a previous study (chapter 2), it was estimated that U. maydis PAL protein has a 80000 subunit size. This is essentially identical to the calculated molecular weight (79277) of the deduced protein. U. maydis PAL, consisting of 724 deduced amino acids, is longer than most known PALs i l l but shorter than the 752 amino acids of pine (Zhang and Chiang, GenBank accession no. U39792), and the 725 amino acids of alfalfa PAL (Gowri et al., 1991). PAL from maize and potato are known to be glycoproteins (Havir, 1973; Shaw et al., 1990). Analysis of PAL gene sequences from alfalfa (Gowri, 1991), bean (Cramer et al., 1989), parsley (Lois et al., 1989), and Ustilago (this study) reveal several potential N-glycosylation sites, but their functional significance remains to be elucidated. Protein data from SDS-PAGE (chapter 2) suggest that posttranslational modification through glycosylation is unlikely in Ustilago PAL, as it migrates at a molecular weight corresponding to the size of deduced protein. Heterologous expression in E. coli of PAL genes from yeast (Orum and Rasmussen, 1992) and parsley (Shultz et al., 1989; Appert et al., 1994) has demonstrated that glycosylation is not essential for the catalytic activity of the enzyme. However, it has been postulated that glycosylation may contribute to enzyme stability, to the correct localization of the enzyme within the cell, and to positioning of the active sites for optimal activity (Havir, 1973; Shaw et al., 1990). Bolwell (1992) reported a role for phosphorylation in the regulation of PAL from bean. Detection of a number of phosphorylation sites in the U. maydis PAL sequence provides the possibility that phosphorylation may have a role in modulating the Usilago PAL protein activity. The observation of a synergistic effect of cAMP with tryptophan on PAL induction in U. maydis cell cultures (chapter 4), and the presence of a cAMP-dependent protein kinase phosphorylation site in the U. maydis PAL sequence, support this possibility, but the process and mechanisms of any such phosphorylation remain unknown. 112 In contrast to most plant PAL genes or yeast PAL genes, no A+C- or C+T- rich sequences are found in the upstream region of the Ustilago PAL coding sequence. As in bean (Cramer et al., 1989), parsley (Lois et al., 1989), and rice PALs (Minami et al., 1989; Zhu et al., 1995), a TATA box is present upstream from the translation start site in Ustilago PAL. In contrast, the Rhodosporidium PAL gene has no TATA box (Anson et al., 1987). The higher eukaryotic consensus polyadenylation signal AATAAA in the 3' noncoding region is found in most plant PAL genes and in the Rhodosporidium PAL gene. The absence of this sequence in Ustilago PAL genes is one of the structural features which distinguishes the Ustilago PAL gene from other known PAL genes. Amino acid sequence identities of 39-40% with yeast fungal PALs and 23-26% with plant PALs (Table 3.7) indicate that the Usilago PAL structure has diverged markedly from other PALs. Furthermore, the absence of introns in the Ustilago PAL coding region is in striking contrast to virtually all known PAL genes. Thus far, most PAL-encoding genes in plants contain a single intron in the 5' end of the gene; the exception is an Arabidopsis gene, PAL3 (Wanner et al., 1995), which contains a second intron further downstream. The tendency in other fungi seems to be the retention of numerous introns, since the R. rubra PAL gene has five introns (Filpula et al., 1988) while that of R. toruloides has six (Anson et al., 1987). Interestingly, as in the case of Ustilago, there is apparently no intron in PAL genes in conifers, Pinus banksiana (Campell, 1991; S. Butland, personal communication) and Pinus taeda (Sederoff etal., 1994). PAL is encoded by a small gene family in most plants, with the exceptions of the large PAL gene family (40-50 genes) in potato (Joos and Hahlbrock, 1992) and the single PAL gene in Pinus taeda (Whetton and Sederoff, 1992). In fungi, the only report available is 113 from R. toruloides, which possesses a single copy of the PAL gene in its gemome (Gilbert et al., 1985). Genomic Southern blot analysis (Fig. 3.8) indicates that only one PAL gene is present in the U. maydis genome. The substantial difference in gene sequence and organization, and the distinct kinetic (i.e. high Km) and physico-chemical (i.e. high pi) properties (chapter 2), are all consistent with the highly diverged Ustilago PAL gene appearing as a distinct branch in the PAL dendrogram (Fig. 3.11). These unique features of U. maydis PAL raise the interesting question of how these differences might influence the function and regulation of PAL activity in Ustilago. Examination of the patterns of conserved sequences between all the HALs and PALs analyzed in this study showed that sequence conservation is restricted to relatively few regions within the protein, consistent with the analysis of Taylor er al. (1990). Given that both proteins catalyze a similar reaction, albeit with different substrates, it seems likely that these conserved regions are important for establishing the architecture and function of a dehydroalanine-containing catalytic centre. Regions of sequence divergence, on the other hand, would be predicted to reflect the ability of the two enzymes to utilize different substrates, as well as differing patterns of regulation and cellular organization. I have confirmed (using P. fluorescens HAL, U. maydis PAL and poplar PAL) that there is no evidence of HAL activity associated with PAL, and wee versa (data not shown). It thus appears that modern HAL and PAL genes share a common ammonia-lyase ancestor, but have diverged extensively to serve very different functions in different organisms, or within the same organisms, as shown in the dendrogram of Fig. 3.11. Unfortunately, information on HAL in plants and fungi is very limited, as is sequence data for PAL in Streptomyces and the true fungi. A more detailed evolutionary study of these two 114 enzymes will require cDNA sequence data from more taxa, particularly in the fungi and prokaryotes. Immunological comparison of proteins can provide useful indications of tertiary and quaternary structural relatedness between proteins from different sources. The patterns of weak recognition (Fig. 3.12) between the U. maydis fungal PAL antibodies and plant PALs, and between plant PAL antibodies and fungal PALs, are consistent with the divergence observed between fungal PAL and plant PAL protein sequences (Fig. 3.11). In addition, the weak detection of yeast fungal PAL by Ustilago PAL antibodies correlates with the extensive protein sequence divergence between these two fungi, as shown in Fig. 3.11. U. maydis PAL and Pseudomonas HAL differ greatly in their properties (e.g. Ustilago PAL has a subunit molecular mass of 80 kDa, whereas the Pseudomonas HAL subunit has a mass of 55 kDa). However, the strong binding of the Ustilago PAL antibodies to Pseudomonas HAL implies that both enzymes possess common epitopes. Their sequence homology (25% identity in protein sequence), and the presence of the most hydrophilic residues closer to the carboxy terminus location in their hydrophilicity profiles (Fig. 3.12. D and E) of the deduced amino acid sequences, also suggest that there could be an unexpected level of structural relatedness between the Ustilago PAL and Pseudomonas HAL. Significant cross-reactivity between the three PAL antibodies tested and the bacterial HAL confirms that the two enzymes are immunologically related. Curiously, however, cross-reactivity was not observed between antibodies raised against bacterial HAL (from either Pseudomonas putida or P. fluorescens) and PAL proteins from 115 Ustilago, yeast, poplar or potato. The reason for this lack of reciprocity is unknown, and may require identification of the main epitopes on both groups of proteins. There has been little success to date in raising stable monoclonal antibodies to PAL proteins, for reasons that are not clear. The immunological enzyme inhibition tests corroborated the presence of immunological relationships between PAL and HAL enzymes detected by Western blot analysis. Again, in this test system, while the anti-bacterial HAL antiserum did not inhibit any of the plant or yeast PALs, it did react with Ustilago PAL, providing further evidence for surprising structural relatedness between this Ustilago PAL and Pseudomonas HAL. None of the anti-PAL or anti-HAL antibodies tested were able to inhibit yeast PAL activity, consistent with the other data showing that yeast PAL is immunologically distinct from PAL of Ustilago and higher plants. To extend the array of antibody probes available for these immunological comparisons, antibodies generated against the mammalian class of HAL would be very useful. Considering the very weak cross-reactivity that the Ustilago PAL protein displays with plant PALs, and even with yeast PAL, these results imply that it may be quite distinct from all other known PALs. This conclusion is supported by the sequencing of the U. maydis PAL gene, which revealed very low sequence homology with other PAL genes. This likely accounts for the failure to clone the U. maydis PAL gene from a U. maydis cDNA library using either plant PAL cDNA or yeast PAL DNA as heterologous probes. Together, the DNA sequence data and the immuno cross-reactivity data should help define those common structural features of PAL and HAL that have been retained during their long period of divergence. 116 To date, PAL genes have not been cloned from plant pathogens and even information on the properties of PAL from plant pathogens is extremely limited. With the cloning of PAL gene from the corn smut pathogen, U. maydis, in this study, and from its host plant, corn (Rosier et al., 1997), the tools are now available to explore the role and the functional divergence of both PALs in the metabolic and physiological context of this plant-pathogen interaction. 117 CHAPTER FOUR Regulation of Phenylalanine Ammonia-lyase by L-Tryptophan In Ustilago maydis 4.1 INTRODUCTION Flow of materials through metabolic networks is effected by controlling the presence of enzymes or by controlling the activities of these enzymes. Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), the first enzyme in the major catabolic pathway of L-phenylalanine in plants and some microorganisms, catalyzes the conversion of L-phenylalanine to trans-cinnamic acid. In plants, this reaction forms the initial step of the phenylpropanoid pathway, a major secondary metabolic pathway that provides precursors for a number of unique and important metabolites (Hanson and Havir, 1981). PAL thus controls the channeling of a substantial portion of carbon from primary into secondary metabolism in plants (Jones, 1984). Plant PAL activity is induced by pathogen attack, and by environmental stresses such as wounding and light (Hahlbrock and Scheel, 1989). The enzyme activity is modulated during plant development in different tissues (Camm and Towers, 1973; Wanner, 1995), in response to hormones (Hughes et al., 1990; Reid et al., 1972; Ward et al., 1989) and in response to heavy metals (Ohl et al., 1990; Preisig et al., 1991). In gherkin and mustard, PAL induction is reported to result from the activation of a constitutive pool of inactive enzyme (Attrige et al., 1974), but in other plant species, PAL induction is the result of de novo synthesis of the enzyme (Hahlbrock and Schroder, 1975), or of a decrease in the rate of degradation of the enzyme (Zucker, 1971). PAL 118 activity is regulated, at least in part, at the level of transcription (Lawton and Lamb, 1987), but can also be regulated by the protein components of a system that inactivates the enzyme in apple skin (Tan, 1980), gherkin hypocotyls (French and Smith, 1975), lettuce (Ritenour and Saltveit, 1996), potato tuber (Zuker, 1968), sunflower leaves (Gupta and Creasy, 1984), and sweet potato roots (Tanaka, 1977). Studies on PAL induction and regulation in microorganisms have been limited to the fungi Rhodosporidium toruloides (formerly Rhodotorula glutinis), Rhizoctonia solani and Neurospora crassa, although phenylalanine catabolism is reported in many microbial species. In R. toruloides, the enzyme is induced by L-phenylanine or L-tyrosine, and growth with glucose or ammonium salts represses enzyme synthesis (Gilbert and Tully, 1982). This regulation of PAL activity by phenylalanine, ammonia, and glucose has been shown to occur at the level of transcription (Gilbert et al., 1983). R. toruloides PAL is also inducible in response either to a signal of nitrogen deprivation or to a signal of carbon limitation (Marusich et al., 1982). In N. crassa, PAL synthesis requires specific induction by L-phenylalanine and nitrogen derepression (Sikora and Marzluf, 1982). R. solani PAL synthesis requires induction by L-phenylalanine, L-tyrosine or L-tryptophan (Kalghatgi and Subba Rao, 1976). The properties and genetic control of PAL in both plants and fungi are important factors in our general understanding of the metabolic processes that yield phenylpropanoid secondary metabolites and energy. The substrate inducibility of fungal PAL activity, and the very different symbiotic or parasitic microenvironments in which fungi exist, raise the 119 question of how the regulation of PAL differs among fungi, and between plants and fungi, and how these differences might be reflected in the properties of the PAL protein. The PAL protein of Ustilago maydis has been characterized and its gene structure described in Chapters 2 and 3, respectively. In this chapter I describe the distinct pattern of regulation of Ustilago PAL, which is very different from the patterns known for plants and other fungi. The Ustilago PAL gene is expressed constitutively, but the enzyme activity can be induced by L-tryptophan. The metabolic fate of L-phenylalanine in this fungus has also been briefly examined. 4.2 MATERIALS AND METHODS 4.2.1 Fungal strains and cultures Ustilago strains used in this study are listed in Table 4.1. Cultures were grown on potato dextrose agar (PDA, Difco) and on complete (Holliday, 1974) or basal medium (2% glucose, 20 mM NH4N03, and salt solution (Holliday, 1974), pH 7.0). A sporidial suspension (1 ml) (Absorbance >2.2 at 600 nm) was inoculated into 50 ml complete or basal medium in 250 ml Erlenmeyer flasks and grown for 24-48 hr at 30°C with shaking (250 rpm). Alternatively, 100 \i\ sporidial suspension was inoculated into 5 ml complete or basal medium in 15 ml culture tubes and grown for 16-36 hr. Unless otherwise indicated, U. maydis strain 518 and basal medium were used for the experiments on PAL 120 Table 4.1. Ustilago species and U. maydis strains used in this study Species/Strain Relevant Genotype U. maydis 518 a2b2 U. maydis 521 a1b1 U. maydis UM031 a1b2 U. maydis UM032 a2b1 U. maydis D132 a1/a2b1/b2 U. maydis D132-9 a1/a2 b2/b1.:HygB r U. maydis uad a2b2 uad U. maydis ubd a2b2 ubd U. maydis adrl a2b2 adrl U. hordei MAT1 U. nigra wild type field isolate U. aegilopsidis wild type field isolate Sporisorium reilianum wild type field isolate 121 regulation. Fungal cell growth was measured by counting the number of sporidia using a haemocytometer, with five replicates. Mesurements for cell growth are illustrated in Figures with column (average value) and vertical bar (± standard deviation). 4.2.2 Chemicals [a-32P]-dATP (3000 Ci/mmol) and L-[3H]-amino acid mixtures were purchased from ICN Biomedical Pharmaceuticals, Inc.. L-[U-14C]-phenylalanine (474 mCi/mmol) was purchased from Amersham International. Hyamine hydroxide was obtained from Fluka G. Laboratories. Diethyl pyrocarbonate (DEPC), cyclic adenosine 5'-triphosphate (cAMP), cycloheximide, actinomycin D, daunomycin, a-amanitin, protein A-Sepharose beads, bovine serum albumin, L-phenylalanine, L-tyrosine, L- or D- or DL-tryptophan, rrans-cinnamic acid, para-coumaric acid, benzoic acid, 4-hydroxybenzoic acid, other phenolics, indole, and indolic derivatives were obtained from Sigma. Westran membrane was purchased from Schleicher & Schuell; Zeta-Probe membrane, from Bio-Rad, and microbial media, from Difco. All other chemicals (analytical reagent grade) used were obtained from Sigma or Aldrich. 4.2.3 Enzyme extraction and assay Cultures were harvested in a microfuge tube and the fungal cells were washed two times with sterile distilled water. The cell pellet was homogenized for 20 seconds at 4°C with alumina and a mini-pestle (Mandel Scientific Co.). The homogenate was suspended 122 in 200 u.1 ice-cold 0.5 M sodium phosphate buffer, pH 8.0, vortexed for 5 min with frequent cooling in ice and then centrifuged at 14,000 x g for 15 min at 4°C. The supernatant was desalted (Pharmacia PD-10 column), and used for PAL assays and other experimental analyses. Unless otherwise described, all experiments were repeated at least twice with three or five replicates. All values illustrated in Tables and Figures are the average + standard deviation of all mesurements (in Figures, column indicates average and vertical bar indicates ± standard deviation). PAL activity (three replicates per protein extract) was measured by a radiometric method as described by Campbell and Ellis (1992) using L-[U-14C]-phenylalanine as substrate. Protein was determined by the Bradford method (Bradford, 1976) with bovine serum albumin as a standard. 4 . 2 . 4 Immuno-blot analysis Enzyme extracts previously described were separated on 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gels as described by Laemmli (1970) and electro-transferred to PVDF membrane. PAL proteins on the blot were detected using U. maydis PAL antiserum as described in Chapter 2. 4.2.5 Immunoprecipitation of radiolabeled PAL protein U. maydis cells were grown in basal medium (50 ml) for 12 or 16 hr with or without L-tryptophan (0.3 mM), supplemented with 50 uCi [3H]-radiolabeled amino acids mixture, and further incubated for 4 hr. Following harvest by centrifugation at 6000 x g for 10 min 123 at 4 °C, the cells were washed three times with sterile distilled water and frozen in liquid nitrogen. Protein crude extracts were prepared by grinding the cells with alumina as described in Chapter 2. Radiolabeled PAL proteins were immunoprecipitated with U. maydis PAL antiserum (or preimmune serum) and protein A-Sepharose 4B beads , using a protocol described in 'Antibodies: A Laboratory Manual' pp. 466-468, (Harlow and Lane, 1988). After heating in SDS sample buffer at 85°C for 10 min, immunoprecipitated proteins were separated from the beads by centrifugation and the radioactivity was measured using a Beckman LS-5000TA scintillation counter. 4.2.6 Phenylalanine catabolism analysis U. maydis cells were grown for 16 hr in complete medium (50 ml) which was then supplemented with 5 nCi L-[U-14C]-phenylalanine (sp. act. 474 mCi/mmol) or 2 \iC\ L-[U-14C]-cinnamic acid (sp. act. 474 mCi/mmol). After two hours incubation, the cultures were separated into medium and cells by centrifugation at 14, 000 x g for 15 min at 4°C. Cell extracts were prepared by boiling in 95% ethanol for 10 min, while the culture medium was extracted with diethyl ether after acidification with 6N HCI. Both solvent extracts were concentrated with a stream of gaseous nitrogen and chromatographed by TLC (1st dimension; n-butanol:ethanol:water = 4:1:1, 2nd dimension; toluene:acetic acid = 4:1), and autoradiographed on X-Omat AR film (Kodak). The metabolites produced from phenylalanine were identified on the basis of their Rf values and co-chromatography with authentic samples. 1 4 C0 2 produced by U. maydis cultures growing in the presence of L-[U-14C]-phenylalanine [0.25 nCi] was captured in Hyamine-hydroxide solution and the radioactivity was measured by liquid scintillation counting. 124 4.3 RESULTS 4.3.1 Induction experiments In order to determine whether PAL activity is detectable in U. maydis cells grown in standard microbial growth media, several nutrient sources were tested. PAL activity was present in cells grown with all the nutrient sources tested, although the level varied (Fig. 4.1). Potato dextrose broth and casamino acids supported higher levels of PAL activity, as well as increased cell growth. Beef extract, on the other hand, supported cell growth but yielded low levels of PAL production in this study. Based on the results shown in Fig. 4.1, regulation of PAL was investigated in two media: a nutrient-rich (complete) medium and a minimal (basal) medium. Depending on inoculum density and freshness, the specific PAL activity levels measured at the early stationary phase of cell growth ranged from 4 to 16 pkatal/mg protein in basal medium, and from 14 to 22 pkatal/mg protein in complete medium. Since aromatic amino acids have been shown to function as inducers of PAL in some fungi (Kalghatgi and Subba Rao, 1976; Gilbert and Tully, 1982; Sikora and Marzluf, 1982), L-phenylalanine, L-tyrosine, and L-tryptophan were tested for their ablity to induce PAL in U. maydis 518 strain. The PAL activity levels obtained from fungal cells grown in 1% glucose media (pH 7.0) with these amino acids (1 mM each) are presented in Fig. 4.2A. The highest PAL activity occurred in cells grown in L-tryptophan-supplemented medium, whereas, L-tyrosine and L-phenylalanine supplementation had very little effect. 125 • PAL Activity H3Cell Growth AN BE CA PD PT Media TT YE Fig. 4.1. U. maydis cell growth and PAL activity in different media. Cells were grown for 24 hr in each medium of 2% AN: ammonium nitrate, BE: beef extract, CA: casamino acids, PD: potato dextrose broth, PT: peptone, TT: tryptone, and YE: yeast extract. 126 Q. O) o E § Q. O >—• o I _l Tip Tyr Phe Phe + Trp No treatment Amino Acids B 2% Phenylalanine 2% Tryptophan Medium Fig. 4.2A. Effect of aromatic amino acids on PAL induction in U. maydis. 1 mM of each amino acid was added into 1 % glucose media and the fungal cells were grown for 36 hr, harvested, and assayed for PAL activity. Fig. 4.2B. Cell growth and PAL activity of U. maydis grown on tryptophan or phenylalanine as sole carbone and nitrogen sources. The fungal cells were grown for five days in 2% tryptophan- or 2% phenylalanine-only medium containing inorganic salts (Holliday, 1974), and harvested for PAL assay and cell count. 127 Interestingly, PAL induction by L-tryptophan was strongly reduced by co-supplementation with L-phenylalanine. The fungus was able to grow slowly on either tryptophan or phenylalanine (2% wt/v) as a sole carbon and nitrogen source (Fig. 4.2B). In this case, culture growth was better in tryptophan medium, but PAL activity was higher in phenylalanine medium. In absolute terms, however, these culture conditions did not result in any marked enhancement of PAL specific activity as compared to the results shown in Fig. 4.2A. To determine whether PAL induction by tryptophan is dependent upon its concentration, different amounts (0.03, 0.1, 0.3, 1.0, and 3.0 mM) of L-tryptophan were used to supplement basal medium cultures. The increase in PAL activity responded linearly to the increase in L-tryptophan concentration up to 0.3 mM amino acid, and then declined (Fig. 4.3). The level of L-tryptophan (0.3 mM) giving the highest PAL induction in U. maydis is substantially lower than the level of L-phenylalanine supplemention 15 mM or 6 mM required for PAL induction in R. toruloides (Marusich and Zamir, 1981; Wick and Willis, 1982). Based on these results, 0.3 mM L-tryptophan was used for the following PAL regulation study. 4.3.2 PAL induction period In order to define the time point of highest PAL inducibility during the culture cycle, Ustilago cultures were supplemented with 0.3 mM L-tryptophan at various time points in the growth cycle, incubated for two hours, harvested and assayed for PAL activity. 128 50 r -P 40 • 30 & 20 • 10 T 0.03 0.1 0.3 1 L-Tryptophan [mM] Fig. 4 .3. Induction of U. maydis PAL at different concentrations of L-tryptophan. L-tryptophan was added 12 hr after inoculation into basal medium. Cells were incubated for 12 hr after adding tryptophan, harvested, and assayed for PAL activity. 129 Enhancement of PAL activity by these additions of exogenous L-tryptophan was only observed in cultures that had entered stationary phase (Fig. 4.4). This pattern was also observed when tryptophan was supplemented at the beginning of culture growth, and cultures were harvested at various time points in the growth cycle and assayed for PAL activity (data not shown). In this case, the elevated PAL levels were stable for at least twelve hours during the stationary phase of culture growth (data not shown). This pattern of highest inducibility and longer stability of Ustilago PAL during the stationary phase of growth differs slightly from R. toruloides PAL, which was highly induced during mid- to late log phase of growth and showed variation in stability during the stationary phase of growth (Wick and Willis, 1982; Gilbert et al., 1983). 4.3.3 PAL inducibility by tryptophan optical isomers, precursors, and metabolites An array of tryptophan optical isomers, precursors, and metabolites was tested for their abilty to induce PAL. All three isomeric forms of tryptophan (L, D, and DL) tested were able to induce higher levels of PAL, and to a similar degree (Fig. 4.5). On the other hand, PAL activity failed to increase in response to the various tryptophan precursors and metabolites tested, although indole-3-pyruvic acid gave a slight positive response. Therefore, PAL induction appears to be tryptophan-specific (Table. 4.2). lndole-3-acetic acid (IAA), which has been reported to occur naturally in U. maydis cultures (Navarre, 1990), reduced PAL activity almost 50% below control levels, while, indole (0.3 mM) itself was inhibitory to fungal cell growth, resulting in the failure of that component of the induction study. 130 60 r 50 • 40 30 20 10 - • PAL Activity With Trp o PAL Activity Without Trp Hi—Cell Growth With Trp -a—Cell Growth Without Trp 10 8 E 3 T) 0) £ 6 -I X tn 75 H 4 S £ s CD ^ 2 a> O 4 8 12 16 24 36 Time (hr) of addition of L-tryptophan Fig. 4.4 Effect of tryptophan addition on cell growth and PAL induction of U. maydis. 0.3 mM L-tryptophan was added at different times in the cell cycle. Cells were harvested 2 hr later and assayed for PAL activity. 131 .£ 60 0 | 50 O) 1 40 CO 3 30 I* 20 o < -J < Q_ 10 0 No Tip DTrp L-Trp DL-Trp Tryptophan Isomers Fig. 4 .5 . Comparison of PAL inducibility in U. maydis by different optical isomers of tryptophan. 0.3 mM of each tryptophan isomer was added to 1% glucose media and the fungal cells were grown for 36 hr, harvested, and assayed for PAL activity. 132 4 .2. Effect of tryptophan analogs and tryptophan-related metabolites on PAL induction in U. maydis ' Compounds Relative PAL Activity (%) b No supplement 100 Anthranilic acid 110 ± 10 lndole-3-acetamide 82 ± 9 lndole-3-acetic acid 55 ± 6 lndole-3-acetonitril 107 ± 11 lndole-3-butyric acid 114 ± 12 lndole-3-carbinol 1 1 6 ± 9 lndole-3-carboxylic acid 101 ± 10 DL-lndole-3-lactic acid 88 ± 7 lndole-3-pyruvic acid 135 ± 5 Kynurenic acid 76 ± 9 Tryptamine 98 ± 10 Tryptophol 86 ± 8 L-Tryptophan 310 ± 12 9 Cells were grown in basal medium for 16 hr before addition of the exogenous compounds (0.3 mM each), harvested after 2 hr further incubation, and used for PAL assay. * Relative PAL acitivity is presented as the percentage of the value of PAL activity in control which has no supplement of the compounds. Control showed 12 (pkat/mg protein) in PAL activity. 133 4 . 3 . 4 Tryptophan effect on PAL induction in other U. maydis strains and Ustilago species The above observation that PAL induction was tryptophan-specific, prompted me to investigate whether PAL induction by L-tryptophan is a common phenomenon in Ustilago fungi. For this purpose, several U. maydis strains and Ustilago species were tested. PAL induction by L-tryptophan was observed in all the U. maydis strains tested (Fig. 4.6). However, among the other Ustilago species, only U. nigra revealed PAL induction by L-tryptophan (Fig. 4.7). All the Ustilago species tested did possess PAL, and its expression was detectable even without L-tryptophan supplementation. These results show that although PAL is widespread among Ustilago species, its induction by L-tryptophan is confined to certain species. 4.3.5 Effect of carbon and nitrogen sources on PAL activity induction Various nutritional carbon and nitrogen sources were tested to examine their effect on PAL activity. Different levels of PAL activity and cell growth were obtained from the cells grown with the carbon sources tested (Fig. 4.8). Among these carbon sources, growth on lactose and galactose resulted in the highest PAL activity, even though they proved to be the poorest sources for cell growth. In general, un-induced U. maydis cultures displayed higher PAL activity when they were grown on carbon sources that supported poorer growth, and lower activity when grown on more readily metabolized sugars such as glucose, mannose and sucrose. 134 50 r U. maydis Strains Fig. 4.6. Effect of L-tryptophan supplement on PAL induction in different U. maydis strains. 0.3 mM L-tryptophan was added into basal media and the fungal strains were grown for 24 hr, harvested, and assayed for PAL activity. 135 60 •Without Trp => Fungus Fig. 4.7. Effect of L-tryptophan supplement on PAL induction in different Ustilago species. The fungal cells were grown in basal media for 36 hr with or without 0.3 mM L-tryptophan, harvested, and assayed for PAL activity. 136 c CD o CL o> E CO CL o < _l < D_ 70 60 50 40 30 20 10 • PAL Activity mce\\ Growth T - 12 20 16 Fru Gal Glu Man Lac Mal Sue Ca MA Sa Carbon Source E T3 <D E x o o i— O O Fig. 4.8. Effect of various carbon sources on U. maydis cell growth and PAL activity. Fru: fructose, Gal: galactose, Glu: glucose, Man: Manose, Lac: lactose, Mal: maltose, Sue: sucrose, CA: citric acid, MA: malic acid, and SA: succinic acid. Cells were grown in 20 mM NH4NO3 media for 36 hr with each carbon source and harvested for PAL assay and cell count. 137 To understand how carbon sources influence PAL activity under tryptophan-induction conditions, glucose was selected as a representative carbon source and its effect on PAL induction in U. maydis was investigated. Glucose supplementation repressed PAL production both in the presence and absence of L-tryptophan, and in both the nutrient rich (complete) and minimal (glutamine only) media (Fig. 4.9A). The repressive effect of glucose was observed under tryptophan-induced conditions if the glucose was present from the beginning of the culture cycle, but not if the extra glucose was added later (16 hr) in the culture cycle (Fig. 4.9B). These results indicate that glucose repression can modulate Ustilago PAL expression, but that the repression effect is limited to the early growth stage. The effects of nitrogen source on U. maydis cell growth and PAL activity level are shown in Fig. 4.10. Provision of nitrogen in the form of inorganic salts, or asparagine, reduced PAL activity far below the levels obtained when L-tryptophan or L-phenylalanine was provided as the sole nitrogen source for cultures. For cell growth, peptone was the best source and no significant difference was found among other nitrogen sources. Varying the ratio of ammonium nitrogen to nitrate nitrogen from 1:10 to 10:1 in the non-amino acid media did influence the level of PAL in the cultures after 24 hr growth (Fig. 4.11), but the pattern of change did not suggest a strong preference for one form of inorganic nitrogen. 4.3.6 Effect of inhibitors and cAMP on PAL activity in vivo 138 No treatment Glucose Tryptophan Tryptophan + Glucose Treatment of media Ohr 16 hr Time of glucose addition Fig. 4.9A. Effect of glucose on U. maydis PAL induction by L-tryptophan. Complete media and glutamine (20 mM)-only media were treated with 2% glucose or 0.3 mM tryptophan or both. The fungal cells were grown in these media for 36 hr, harvested, and assayed for PAL activity. Fig. 4.9B. Effect of time of glucose addition on U. maydis PAL induction. An additional 3% glucose was added to L-tryptophan-supplemented basal media at the beginning (0 hr) or at the mid log phase (16 hr) of cell growth. The fungal cultures were incubated for a total of 36 hr, harvested, and assayed for PAL activity. 139 60 r :ein) 50 o Q. 40 E CO ( Pk 30 >> > -4—' O 20 < < D_ 10 o I-•PAL Activity mCeW Growth rSft NS NP NN TRP PHE ASP PT Nitrogen Source 14 _ E 12 % CD E 10 1 8 2 x <n 6 "CD JO 4 % o c5 2 = CD O 0 Fig. 4.10. Effect of nitrogen source on U. maydis cell growth and PAL activity. 0.3 mM of each nitrogen source was added to 2 % glucose media and the fungal cells were grown for 36 hr and harvested for PAL assay and cell count. NS: (NH4)2S04, NP: NH4H2P04, NN: N H 4 N O 3 , TRP: tryptophan, PHE: phenylalanine, ASP: asparagine, and PT: peptone. 140 20 r 1:10 1:3 1:1 3:1 Ratio of NH 4:N0 3 10:1 Fig. 4.11. Effect of the ratio of NH4:N03 on PAL activity in U. maydis. The fungal cells were grown for 36 hr in 2% glucose media supplemented with various ratios of ammonium to nitrate nitrogen (total 0.3 mM), harvested and assayed for PAL activity. 141 It had previously been determined that purified U. maydis PAL could be partially inhibited by the reaction product, f-cinnamic acid, and strongly inhibited by the synthetic inhibitor, AIP (Chapter 2). When these compounds were added to the L-tryptophan-supplemented culture medium at appropriate concentrations, they were found to have differing effects on growth and extractable PAL activity (Fig. 4.12). Cinnamic acid substantially inhibited culture growth, but had little effect on the levels of PAL induced by 0.3 mM L-tryptophan supplementation. Addition of AIP, on the other hand, completely abolished PAL activity in the cell extracts but had no effect on culture growth over 24 hr. Interestingly, addition of cAMP (1 mM) produced a modest enhancment of PAL induction but did not affect culture growth. With this observation, further studies were undertaken to gain insight into the possible invovement of the cAMP cascade in the regulation of PAL. For this, a U. maydis adenylate cyclase deficient mutant (uad, Gold et al., 1994a), and protein kinase A (PKA) deficient mutants adrl (deficient in a catalytic subunit, Orth et al., 1995) and ubd (deficient in the regulatory subunit, Gold et al., 1994a), were tested in L-tryptophan-supplemented growth conditions for their PAL induction respose (Fig. 4.13). PAL was induced to similar levels in the adenyl cyclase mutant and in wild-type U. maydis, but the PAL induction was reduced by 20-30% in the PKA mutants. 4.3.7 Influence of physical environments In order to examine the effect of environmental factors on PAL regulation, U. maydis cultures were grown in complete medium under various light, pH, and temperature 142 • P A L activity ElCell growth Water AIP t-Cinnamic acid Compounds cAMP Fig. 4.12. Effect of cAMP (1 mM), AIP (100 yM), and f-cinnamic acid (0.3 mM) on cell growth and PAL activity of U. maydis. Compounds were added to basal medium, and the fungal cells were grown for 36 hr and harvested for PAL assay and cell count. 143 50 r ^ 40 • •Without Trp ®With Trp 30 -£ 20 • 10 • Wild type uad ubd U. maydis mutants adrl Fig. 4.13. Effect of L-tryptophan supplement on PAL induction in the U. maydis adenylate cyclase deficient (uad) and protein kinase A deficient (ubd, adri) mutants. The mutants were grown in basal media for 36 hr with or without 0.3 mM L-tryptophan, harvested, and assayed for PAL activity. 144 regions. No apparent PAL induction or inhibition was observed when the cultures were incubated in light or dark, or at different medium pH values (pH 4, 5, 6, 7, 8), and/or at different temperatures (18, 24, 30°C) (data not shown). It seems that expression of U. maydis PAL activity is not overtly sensitive to physical environmental factors. 4.3.8 No evidence for o*e novo synthesis in PAL induction Induction of PAL in most plants and some fungi results from de novo synthesis of the enzyme. The origins of induced PAL activity in U. maydis were investigated by the use of inhibitors of translation and transcription, and with radiotracers and immunoprecipitation. In both the L-tryptophan-induced and uninduced conditions, PAL activity was reduced when U. maydis cells were incubated in the presence of actinomycin D, a transcriptional inhibitor, or cycloheximide, an inhibitor of translation (Table 4.3). This implies that gene transcription and protein synthesis are necessary to maintain PAL levels in both conditions during the log phase of culture growth. However, radiolabeling of newly synthesized proteins showed that there were no differences in PAL protein labeling between the cells grown in L-tryptophan-induced and uninduced conditions (Table 4.4). This result was consistent with Western blot results which showed PAL bands of similar intensity in protein samples from L-tryptophan-induced and uninduced cultures (Fig. 4.14). Taken together, these observations suggest that the induction of increased PAL activity by L-tryptophan supplementation does not result from de novo synthesis of PAL, but from activation of the PAL enzyme which has been produced during the log phase of culture growth. The modest inhibition by the actinomycine and cycloheximide treatments may indicate that other proteins needed for PAL activation are turning over in the 145 Table. 4 .3 . Effect of actinomycin D and cycloheximide on U. maydis PAL activity Treatmenta PAL Activity (pkat/mg protein) Induced 0 Uninduced 12hr 16hr 12 hr 16 hr No addition 19.6 + 1.8 32.8 ±2.2 13.3 + 1.6 20.5 ±2.1 Actinomycin D 13.9 ±1.9 23.3 ± 0.8 9.2 ± 1.2 15.1 ±0.6 Cycloheximide 16.2 ±1.7 26.4 ±1.3 11.3 ± 1.0 16.8 ±1.2 a Actinomycin D (100 u.g/ml) and cycloheximide (10 u.g/ml) were added at mid (12 hr) or late (16 hr) log phase in the cell growth cycle, and cells were harvested 4 hr later and used for PAL assay. " Induced: L-tryptophan (0.3 mM) was supplemented to basal medium at the time of inoculation, uninduced: no L-tryptophan was supplemented. 146 Table 4 . 4 . PAL protein synthesis in L-tryptophan-induced and -uninduced conditions0 Cell harvest Newly synthesized PAL (dpm)6 time Induced Uninduced 16 hr 382 ± 36 388 ± 47 20 hr 461 ± 56 473 ± 62 a Induced: L-tryptophan (0.3 mM) was supplemented to basal medium at the time of inoculation, uninduced: no L-tryptophan was supplemented. b Proteins of U. maydis were radio-labelled in vivo with L-r^ H]-amino acid mixture. The labelled PAL proteins were immunoprecipitated with anti-PAL antibodies and counted. 147 kDa 205 — 116— 97-66 — 4 3 — <PAL Fig. 4.14. Immunoblot detection of PAL production in U. maydis grown in L-tryptophan-induced and -uninduced conditions. Protein samples (20 u.g) extracted from the cultures grown for 36 hr in 0.3 mM L-tryptophan-supplemented (lane 1) or unsupplemented (lane 2) basal media, were separated in a 7.5 % SDS-PAGE gel, transferred onto a PVDF membrane and immuno-detected with U. maydis PAL antibodies using the NBT/BCIP visualization system. 148 cells.The fact that the greatest enhancement of PAL activity by exogenous L-tryptophan is obtained in the stationary phase of culture growth (Fig. 4.4), when most protein synthesis has ceased, is consistent with this interpretation. 4.3.9 Metabolic fate of L-phenylalanine in U. maydis cultures Chromatographic analysis of the soluble products of metabolism of L-14C-phenylalanine 14 or C-f-cinnamic acid after 2 hr incubation detected radioactive label at positions on the chromatograms corresponding to r-cinnamic acid, benzoic acid, and 4-hydroxybenzoic acid, and possibly other hydroxybenzoic acid derivatives (Fig. 4.15). These products were detected in the culture medium as well as in the cell extracts. Incubation of the U. 14 14 maydis cultures with L- C-phenylalanine resulted in a slow release of C0 2 over a 6 hr period, but this amounted to less than 0.2% of the potential label available if the phenylalanine were to be totally catabolized. No phenylalanine transaminase activity could be detected in cell extracts, whereas 4-hydroxycinnamyl Co A-ligase activity is detectable that is active with f-cinnamic acid but not with 4-hydroxycinnamic acid as substrate. Taking these observations together, a plausible catabolic pathway for phenylalanine in U. maydis could be formulated as shown in Fig. 4.16. 149 Fig. 4.15. Autoradiogram of the radioactive metabolites recovered from U. 14 14 maydis growing in L-[U- C] phenylalanine or [U- C]f-cinnamic acid. A and B indicate autoradiograms of cell extract and medium extract, respectively, from the cultures grown in the presence of 14C-phenylalanine. C and D indicate autoradiograms of cell extract and medium extract, respectively from the cultures grown in the presence of 14C-f-cinnamic acid. The developed TLC plates were exposed to X-ray film for one week. CA: r-cinnamic acid, BA: benzoic acid, 4BA: 4-hydroxybenzoic acid, and PHBA: possible hydroxybenzoic acid derivatives. 150 L-Phenylalanine I PAL Cinnamic acid derivatives <= frans-Cinnamic acid I 4CL I beta oxidation Benzoic acid I BAHs 4-hydroxybenzoic acid and other hydroxybenzoic acids Possible Melanin Precursors => => C 0 2 (i.e. Catechol Derivatives) Fig. 4.16. Schematic diagram of catabolic pathway of phenylalanine in U. maydis. PAL:phenylalanine ammonia-lyase, 4CL:4-coumaryl Co A ligase, BAHs:benzoic aicd hydroxylases. Arrows indicate identified way (I) and predicted way (II). 151 4.4 DISCUSSION 4.4.1 PAL induction by L-tryptophan The influence of aromatic amino acids on fungal PAL regulation has been studied in only a few species. In the red yeast, R. toruloides, L-phenylalanine and L-tyrosine acted as PAL inducers (Marusich et al., 1981; Gilbert et al., 1983), while in N. crassa, L-phenylalanine (Sikora and Marzluf, 1982), and in R. solani, L-phenylalanine, L-tyrosine, and L-tryptophan all served as PAL inducers. In all of these cases, the best PAL inducer was L-phenylalanine. In contrast, the most effective inducer of PAL activity in U. maydis was L-tryptophan (Fig. 4.2), although L-phenylalanine had some effect. However, induction of PAL in Ustilago cultures by L-tryptophan does not appear to be a typical microbial catabolic system directed at nutrient scavenging. First, a significant basal level of PAL is always detectable in the cultures, unlike the three fungal species mentioned above, in which PAL activity is detectable only when the fungi are grown in media supplemented with aromatic amino acids which induce PAL. Second, in contrast to other fungi like R. toruloides and A/, crassa, the inducer does not appear to have a logical metabolic connection with the reaction catalyzed by the induced enzyme. L-Phenylalanine, the obvious induction candidate, is much less effective in this role than L-tryptophan. Finally, the L-tryptophan effect on PAL induction is not operative in all the Ustilago species tested. 152 Since PAL induction in U. maydis results only from tryptophan supplementation and not from tryptophan precursors or metabolites, it is worth asking whether the endogenous level of tryptophan in the host plants is sufficient for U. maydis PAL induction. There are no data for corn, but the level of free tryptophan in tobacco leaves and carrot cell cultures has been reported as 16.7 u.M (Widholm, 1972) and 81 u.M (Widholm, 1974), respectively. In this study, Ustilago PAL was measurably induced by 30 u.M tryptophan supplementation (Fig. 4.3), suggesting that Ustilago PAL might be inducible by either the endogenous level of tryptophan or enhanced levels of tryptophan resulting from the Ustilago fungus infection in its host corn plants. 4.4.2 Regulatory features of PAL The production of many inducible enzymes by microorganisms is subject to catabolite repression when the organisms are grown in the presence of a potential carbon and energy source. In addition to carbon catabolic enzymes, many proteins, including those involved in bioluminesence, photosynthesis, sporulation, antibiotic biosynthesis, pigment biosynthesis, and extracellular macromolecular degradation, have been shown to be subject to catabolite repression (Saier, 1991). Glucose repression was observed with U. maydis in the present study (Fig. 4.9), since both basal PAL levels and the induction of PAL by L-tryptophan were reduced by glucose addition to the medium. A similar phenomenon was reported in R. toruloides, where PAL induction by L-phenylalanine was repressed by glucose (Gilbert and Tully, 1982). 153 cAMP is widespread among eukaryotes and prokaryotes and is implicated in numerous regulatory events in fungi, including carbon catabolism, growth, and developmental phenomena (Pall, 1981; Thevelen, 1988; Uno, 1992), and in the regulation of expression of certain fungal enzymes (Herman et al., 1990; Terenzi et al., 1992). With the observation of a stimulatory effect of exogenously supplied cAMP on PAL activity (Fig. 4.12), the possible role of cAMP in the regulation of Ustilago PAL by tryptophan was investigated using an adenylate cyclase deficient mutant (uad) and cAMP-dependent protein kinase A (PKA) deficient mutants (ubd and adrl). Both types of mutants were able to display PAL induction in the presence of exogenous tryptophan (Fig. 4.13), implying that cAMP and PKA are not essential for this phenomenon. However, the levels of PAL in the mutants defective in ubd and adrl were reduced compared to those in the wild type. PKA could, therefore, still be involved in the regulation of PAL induction, either by some pleiotropic effect or perhaps by mediating the phosphorylation of PAL proteins themselves. There are many predicted phosphorylation sites in the amino acid sequence of PAL deduced from the U. maydis PAL gene (Chapter 3), including one cAMP-dependent PKA phosphorylation site. Bolwell (1992) has suggested that the inactivation and turnover of PAL in elicitor-treated bean cells is in part regulated by phosphorylation, which is possibly cAMP-dependent. However, the evidence for occurrence of cAMP in plants is not convincing (Bolwell, 1995). The induction of PAL in U. maydis by tryptophan is not likely to result from de novo synthesis of the protein, as shown by immunoprecipitation (Table 4.3) and Western blot assay results (Fig. 4.14). The alternative mechanisms would appear to be that some form of post-translational protein modification (such as phosphorylation of the enzyme), 154 or a metabolic (non-covalent) influence on the Ustilago PAL activity. This behavior differs from that seen in Rhodosporidium and Neurospora (Gilbert et al., 1985; Sikora and Marzluf, 1982), where induction of elevated PAL activity by phenylalanine is controlled primarily at the transcriptional level. Some aspects of the results obtained in this study are reminiscent of classic microbial secondary metabolism. For example, the tryptophan inducibility of PAL is invoked only during the stationary phase of culture growth, and the induction appears to require tryptophan itself, rather than tryptophan metabolites or precursors. It is noteworthy that IAA is most actively produced at the stationary phase in the culture cycle of U. maydis (Navarre, 1990). Considering that tryptophan is the major precursor for IAA production in this fungus, the induction and suppression of PAL by tryptophan and IAA, respectively, suggests that a metabolic control mechanism links phenylalanine metabolism to tryptophan metabolism during the late growing period of U. maydis. It has been cleady estabilished in both prokaryotes and eukaryotes that the synthesis of these two aromatic amino acids, phenylalanine and tryptophan, is derived from chorismate through the shikimate pathway (Braus, 1991; Hrazdina and Jensen, 1992). Logically, the synthesis and catabolism of each amino acid will be regulated to reflect their demands and their levels. From this perspective, it is conceivable that U. maydis PAL regulation is indirectly under the influence of tryptophan levels through metabolic feedback mechanisms that control synthesis and catabolism of all the aromatic amino acids. Studies on the effect of tryptophan on the synthesis of the PAL substrate, 155 phenylalanine, could provide insight into potential metabolic connections between tryptophan metabolism and PAL regulation. f-Cinnamic acid, the product of the PAL reaction, is moderately inhibitory to U. maydis cell growth. Toxicity of f-cinnamic acid was also observed in R. toruloides cultures (Marusich era/., 1981; Kane and Fiske, 1985). In some plant systems like bean (Bowell et al., 1988) and alfalfa (Orr, 1993) cell suspension cultures, addition of exogenous f-cinnamic acid suppressed PAL induction. This has led to the suggestion that feedback inhibition by f-cinnamic acid could help control diversion of L-phenylalanine from primary metabolism to secondary metabolism and thereby avoid the accumulation of f-cinnamic acid to levels that might become toxic to cells. In U. maydis, however, there was no evidence that exogenous cinnamic acid influenced PAL induction. 4.4.3 Metabolic features of phenylalanine catabolism in the context of the host-pathogen interaction The major metabolites of f-cinnamic acid detectable in the Ustilago cultures are benzoic acid, and possibly, its derivatives, a pattern consistent with degradation of the phenylpropanoid skeleton by p-oxidation of the side-chain and oxidation of the ring. The detection of 4-coumaryl Co A-ligase (4CL) activity that was active not with p-coumaric acid, but with f-cinnamic acid, as a substrate suggests that the biosynthetic route to hydroxybenzoic acids in U. maydis differs somewhat from that of plants, which use p-coumaric acid as a preferred substrate for 4CL. 156 What are the roles of benzoic acid and its derivatives in U. maydis biology? They might serve as precursors for the synthesis of physiological effectors, analogous to signal molecules like salicylate or its derivatives in plants (Malamy et al., 1990). Such roles for hydroxybenzoic acids have not been elucidated in fungi, but the recent finding in tobacco plants (Yalpani et al., 1993) that salicylate is synthesized from phenylalanine via t-cinnamic acid and benzoic acid, provides a possible precedent for a similar process in fungi. Several fungi that contain PAL, including Ustilago hordei, have been reported to metabolize L-phenylalanine into benzoic acid and its derivatives (Towers et al., 1969), and salicylate and its derivatives have been found in the extracts from U. maydis teliospores (Piattelli era/., 1965). 4.4.4 Potential roles of PAL in U. maydis In contrast to higher plants, the role of PAL in fungi is not obvious. Earlier information on fungal PAL regulation was restricted largely to R. toruloides and N. crassa, and no definitive biological functions were uncovered in those studies. It seems reasonable to assume, however, that PAL could provide one mechanism for catabolism of L-phenylalanine acquired during parasitic growth of plant pathogens such as Ustilago, Rhizoctonia or Altemaria, or during saprophytic growth of non-pathogenic fungi (e.g. Rhodosporidium and Neurospora). Those fungal species that possess PAL have been shown to convert exogenous phenylalanine to various phenolic metabolites. It is also conceivable that cinnamic acid is required as a substrate for biosynthesis of some essential physiological effector or protectant for U. maydis, perhaps within a 157 specific developmental context. Derivatives of cinnamic acid which might function as germination self-inhibitors in spores, would be potential candidates, and inhibitors derived from cinnamic acid have been found in urediospores in rust fungi (Macko et. al., 1970; Macko et. al., 1971). Since PAL activity could also be detected in urediospores of the rust fungus (Moerschbacher et al., 1988), it seems likely that PAL would be involved in the biosynthesis of these endogenous inhibitors. Self-inhibition is overcome by nutrients (including peptone or phenylalanine) in Glomerella cingulata (Lingappa and Lingappa, 1965), and is glucose-dependent in Fusarium solani (Griffin, 1970a and b), indicating that this process can be metabolically modulated in some cases. These responses at least superficially resemble the pattern of U. maydis PAL regulation, as reflected in the glucose repression and aromatic amino acid induction results. Since self-inhibition is also operative in teliospore germination of Ustilago fungi (Pritchard and Bell, 1967), Ustilago PAL may thus play a role in the regulation of this self-inhibition. Identification of the chemical nature of the germination self-inhibitors in Ustilago fungi would allow this possibility to be tested. The function of melanin in fungi is still not clear but roles in pathogenicity and virulence (Polak, 1989), resistance against microbial stress such as hydrolytic enzymes (Kuo and Alexander, 1967) and protection from environmental stress (e.g. UV irradiation, Bell and Wheeler, 1986) have all been suggested. Formation of teliospores within the plant in the last stage of the U. maydis infection cycle is accompanied by melanization, which creates a UV-impermeable shield for the exposed spores. Catechol derivatives and salicylic acid have both been identified from plant melanin (Yoshida, 1969), and these phenolics were also reported as components of melanin in U. maydis teliospores (Piattelli 158 et al., 1965). Considering that catechol derivatives and salicylic acid might be synthesized via benzoic acid, and thus from phenylalanine, it is very likely that the biosynthetic route to Ustilago melanin will require PAL activity. Such a route has been suggested for the biosynthesis of melanin in the fungal pathogen Alternaria (Pridham and Woodhead, 1977). Rhodotorula PAL has been postulated to play a role in the synthesis of another pigment which is very effective in protecting cells from UV light (Ogata et al., 1967). Much more browning (melanin-like) pigmentation was observed in U. maydis cells grown in PAL-inducing (tryptophan-supplemented) media than in those grown in PAL-uninducing (tryptophan-unsupplemented) media. It is thus tempting to speculate that, as with plant PALs, a common role for fungal PAL might be formation of aromatic metabolites that enable the fungi to survive environmental stresses. Clarification of the function of PAL in U. maydis, and of the relationship between the PAL reaction and tryptophan metabolism, will probably require a molecular genetic approach. For this, disruption of the U. maydis PAL gene cloned in Chapter 3 and evaluation of the resulting null phenotypes would provide valuable insights into the biological function of this well-known, but little understood, enzyme within the corn smut organism. 159 CHAPTER FIVE Conclusions and Future Directions 5.1 Conclusions For the first time, PAL has been purified to apparent homogeneity, and the corresponding gene cloned from U. maydis, a filamentous fungus and a plant pathogen. Analysis of the purified protein showed that U. maydis PAL resembles other known PALs in many respects, e.g. it is a cytosolic, homotetrameric protein with a molecular mass of 320 kDa, requires no cofactors or no thiol reducing reagents, and is stable in the absence of glycerol. In contrast to yeast PAL (Camm and Towers, 1973), U. maydis PAL was not able to deaminate L-tyrosine. In other respects, the Ustilago PAL is somewhat unusual. It has a relatively high K„, (1.05 mM) for phenylalanine, a high pi (6.3), and notable temperature stability. Ustilago PAL appears to be composed of 724 amino acids and be encoded by a single gene. Analysis of amino acid sequence showed that Ustilago PAL shares low homology with other known PAL genes. This, together with the weak immunological cross-reaction between U. maydis PAL and plant PALs, and between U. maydis PAL and yeast PAL, shows that the structures of Ustilago, yeast, and higher plant PAL proteins have diverged substantially. It is worth noting that no introns are present in Ustilago PAL sequence, a pattern that is quite different from that of angiosperm plants and yeasts. 160 Ustilago PAL shows a modest level of amino acid sequence identity with the other ammonia-lyase, HAL. The present work also demonstrated, for the first time, the immunological relatedness between PAL and HAL. The immunological cross-reactivity between a Pseudomonas bacterial HAL and Ustilago PAL (or poplar and alfalfa PALs) is consistent with the amino acid sequence homology, and must reflect.common structural features in these two ammonia-lyases that have been retained despite the long time since HAL and PAL diverged. The weakness of the cross-reactivity between Ustilago (or yeast) PAL and the plant PALs is consistent with the divergence observed between fungal PAL and plant PAL protein sequences. This may reflect possible divergence of PAL functions in different organisms. The production of PAL only in phenylalanine-supplemented cultures in saprophytic fungal species suggests that their PAL expression may be dependent upon the amount of the phenylalanine available in their environmental niche. This situation differs from that of pathogenic Ustilago fungi which are involved in dynamic interactions with their specific hosts. The endogenous pools of amino acids in hosts are typically small, and may be insufficient to serve as an inductive signal. Ustilago fungi may therefore have adopted a different mode of PAL regulation. Unlike other known fungal PALs, Ustilago PAL can be produced constitutively at low levels by a range of Ustilago fungi when grown in complete media. In contrast to plant PALs, physical environmental factors such as light, pH, and temperature, do not appear to influence the induction and repression of the enzyme activity. Carbon catabolic repression of PAL activity induction is, however, operative in U. maydis. Analyses of growth and PAL activity induction indicate that PAL can be induced to higher level by L-tryptophan during the stationary phase of culture 161 growth. There was no evidence of de novo PAL synthesis during PAL induction time by L-tryptophan, suggesting that post-translational activation mechanism may undedine this phenomenon. The intracellular location of PAL in the biotrophic fungus U. maydis (this study) is in contrast to the intracellular and extracellular distribution of PAL in the saprophytic fungus N. crassa (Sikora and Marzluf, 1982). The secretion of PAL outside the cell can vary between different fungi and perhaps between different developmental states or environmental situation. The possible secretion of PAL by fungi in planta would have important implications and merits further investigation. If U. maydis secretes PAL during or after the penetration of its host cells, it might affect the outcome of the infection process by modifing the host response. 5.2 Future directions The followings suggestions may be useful to guide further studies of the biological role of Ustilago PAL. • Extending the present immunological analysis to include mammalian HAL and if possible plant HAL would provide further insight into the pattern of immunological relatedness between PAL and HAL proteins across different phyla. 162 • One of the most common mechanisms for post-translational modifications of enzyme activity is phosphorylation. Many putative phosphorylation sites can be identified in the deduced U. maydis PAL protein sequence. If PAL induction involves phosphorylation, or if other proteins must become phosphorylated in order to act on the PAL protein, it should be possible to interfere with these processes by use of appropriate inhibitors. It may also be possible to detect incorporation of 3 2P into PAL in radiotracer studies. • A tentative metabolic pathway of phenylalanine degradation has been suggested (Fig. 4.17), based on some preliminary radiotracer experiments. This pathway, combined with earlier reports on the components of melanin (Piattelli et al. 1965), may suggest a link between PAL and the biosynthetic pathway for melanin in U. maydis. Based on our understanding of melanin functions, and other physiological functions of phenolics derived from phenylalanine, PAL could be envisioned to play an important role in development and survival of U. maydis. Examinations of the structures and properties of compounds derived from phenylalanine through PAL in U. maydis would be useful to test this idea. The ultimate test of the essentiality of PAL would be elimination of the PAL function by creating a pal' mutant genotype through gene disruption. In conclusion, the studies described in this thesis have provided new information concerning the properties, structure and regulation of Ustilago PAL, as well as the metabolic fate of L-phenylalanine in this fungus. The availability of Ustilago PAL-antibodies and a full-length PAL gene sequence will provide useful tools for further study of the structure and function of PAL, as a self-assembling, post-translationally modified, multimeric protein. U. maydis PAL is an unusual member of the PAL family, 163 highly diverged from plant and yeast PALs and sharing interesting similarities with HAL. Study of recombinant U. maydis PAL in comparison to both HAL and other PAL species, using X-ray crystallographic analysis and site-directed mutagenesis, would provide definitive insights into the structural features retained by both ammonia-lyases during their evolutionary divergence. 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Plant Physiol. 47:442-444. 184 APPENDIX A Cloning and Sequencing of a cDNA Encoding Aspartate Semialdehyde Dehydrogenase in Ustilago maydis Aspartate semialdehyde dehydrogenase (ASADH) catalyzes a crucial branch point reaction in the microbial amino acid biosynthetic pathway which leads to the production of L-lysine, L-methionine, L-threonine, and L-isoleucine from L-aspartate. The essential nature of the ASADH gene has made it a target for development of new antimicrobial agents (Cirillo etal., 1991). The gene encoding ASADH has therefore been cloned from several pathogenic bacterial species, but the corresponding gene from eukaryotic pathogenic organisms has yet to be cloned. During the studies on the cloning of the gene encoding phenylalanine ammonia-lyase from Ustilago maydis (chapter 3), a ASADH-encoding cDNA clone was isolated while immunoscreening a XZAPR U. maydis cDNA library. Given the novelty of this gene, the full sequence and structural analysis of the ASADH cDNA were conducted. The nucleotide sequence analysis of the 1182-bp EcoR\-Xho\ fragment insert of the cDNA clone revealed that the cDNA contains a 42-bp 5'-untranslated region, the translation start site, a 1098-bp open reading frame encoding a protein of 366 amino acids, a TAA stop codon, and a 39-bp 3'-untranslated region (Fig. 1). The G+C content is 55%. Southern hybridization of U. maydis genomic DNA digested with several different restriction enzymes, using the cloned ASADH cDNA as a probe, suggests that the U. 185 A A T T C C G C A C G A G C C T T G A T C A A C A A G A G C A C G C A T C A T A C G A T G A C G T C T T C T T C C T C A 6 0 M T S S S S 6 C A A C A G A A G C T C A A A G T C G G C C T G C T C G G C G C T A C G G G C A C A G T A G G T C A G C G A T T C A T C 1 2 0 Q Q K L K V G L L G A T G T V G Q R F I 2 6 C T T C A A C T T G C T G A T C A C C C G C A A T T C G A G C T C G C C G C T C T G G G T G C A T C G T C T T C T T C G 1 8 0 L Q L A D H P Q F E L A A L G A S S S S 4 6 G C A G G T A A G C C G T A C T T G G A A G C A G T A C A A G G G A G G T G G A A G C A G A T C C G A A G G G T T C C C 2 4 0 A G K P Y L E A V Q G R W K Q I R R V P 6 6 G A T A A T G T T G C A C A G A T G C C C G T A T A C G A A T G C A A G C C G G A A T A C T T T G C G A G T G C A G T A 3 0 0 D N V A Q M P V Y E C K P E Y F A S A V 8 6 G T C T C T C C G G T C T G G A T T C A G G T C C A G C G G A C C G T C G A G G A C G C T T T C C G T A G A G C C G A G 3 6 0 V S P V W I Q V Q R T V E D A F R R A E 1 0 6 T T G A G A G T G T T T T C C A A T G C A A A G A A C T A C C G T A C C G A T C C A T T G T G C C C A T T G G T G G T G 4 2 0 L R V F S N A K N Y R T D P L C P L V V 1 2 6 C C A C T G G T C A A C C C G G A G C A C A T G G A G A T C T T G C C A T T C C A G A G A C A G C A A G T G G G G A C C 4 8 0 P L V N P E H M E I L P F Q R Q Q V G T 1 4 6 A A G A A G G G A T T C A T C G T T A C C A A C G C C A A C T G C T C T A C C A C G G G C A T C G T C G T A C C G C T G 5 4 0 K K G F I V T N A (jty C | S T T G I V V P L 1 6 6 A A A G C G C T C G A G G C C A A G T T T G G A C C G C T G G A T ^ A A G A T T C T A G T C A A C A C G A T G C A G G C C 6 0 0 K A L E A K F G P L E K I L V N T M Q A 1 8 6 A T T T C G G G T G C T G G T T A C C C T G G A G T T T C T T C G C T C G A C A T C T T G G A C A A C G T T G T G C C A 6 6 0 I S G A G Y P G V S S L D I L D N V V P 2 0 6 T T C A T C A G C G G T G A G G A G G A G A A G A T C G A G T G G G A G A C C G C C A A G A T C T T G G G T G G C A T C 7 2 0 F I S G E E E K I E W E T A K I L G G I 2 2 6 A A A A C G G A C A A A A C C G C T T T T G A C T A C C A T G A A G A G C A C C C A C T C A A G G T T T C G G C G C A C 7 8 0 K T D K T A F D Y H E E H P L K V S A H 2 4 6 T G C A A C C G T G T T C C G G T C A T C G A T G G C C A C A T G G A G T G C G T T T C T G T G T C G T T C A A G A A C 84 0 C N R V P V I D G H M E C V S V S F K N 2 6 6 C G A C C T G C A C C C T C G G T T G A C G A G G T C A A A A A G T G C C T G C A A G G A T T T A C C A C G G A A G C A 9 0 0 R P A P S V D E V K K C L Q G F T T E A 2 8 6 C A G A C C A T C G G C G T T C A C T C G G C T C C T A A G C A G G C C A T T A C G G T G C A C G A G G A G C A G G A C 9 6 0 Q T I G V H S A P K Q A I T V H E E Q D 3 0 6 C G C C C G C A A C C A C G C C T C G A T C G T G A C T G G C A G A A C G G T G C T G G T G T C A A T G T C G G A A G G 1 0 2 0 R P Q P R L D R D W Q N G A G V N V G R 3 2 6 G T A C G C G A A T G T C C C G T G T T T G A C A T C A A G T T T G T C G T G C T C T C G A A C A A T G T C A T G A T C 1 0 8 0 V R E C P V F D I K F V V L S N N V M I 3 4 6 G G T G C T G C T A C C A G C T G G T T C A T G A A C G C A G A G A T C G C G C T C G C C A A G G G T T A C C T T T C G 1 1 4 0 G A A T S W F M N A E I A L A K G Y L S 3 6 6 T A A T C A T C A T G T G T G G T C A C C T G A A A T C G C A T A C T T T T C G C C 1 1 8 2 Fig. 1. The nucleotide sequence and deduced amino acid sequence (upper case) of the U. maydis ASADH cDNA (GenBank accession No. D11111). The active site cysteine residue is boxed, a potential N-glycosylation site is circled, and the stop codon is underlined. A potential mitochondrial transit peptide is predicted at amino acid position 1 to 25. The putative pyridine nucleotide binding region (amino acids 13-41) and substrate binding region (amino acids 240-260) are underlined. 186 maydis ASADH is likely to be encoded by one gene (Fig. 2). PCR amplification of genomic DNA using primers derived from the 5'-terminal and 3'-terminal regions of the open reading frame in the U. maydis ASADH cDNA, produced a band similar in length to the full length ASADH cDNA, and Southern blot analysis verified that the PCR product was derived from ASADH (Fig. 3). This implies that no substantial introns are present in the U. maydis ASADH gene. The predicted isoelectric point and molecular mass of the deduced U. maydis ASADH protein are estimated as pH 7.6 and 40.2 kDa, respectively. The amino acid sequence identity between ASADHs in U. maydis and other species ranged from 53% to 21% (Fig. 4). However, conservation of ASADH amino acid identity between fungal and bacterial species is typically limited to a few regions such as the putative pyrimidine nucleotide binding domain in the N-terminal region, the substrate binding region, and the core region containing the active site (Ouyang and Viola, 1995). Interestingly, the strict conservation among ASADHs in bacteria and fungi of a Gly-XX-Gly-XX-Gly (X is any amino acid) motif in the NADP-binding region (Fig. 4) is also found in the N-terminal region of isoflavone reductase, a NADP-dependent oxidoreductase in plants (Paiva et al., 1994). This pattern distinguishes it from other NADP-dependent oxidoreductases which have either a Gly-X-Gly-XX-Gly motif (homoserine dehydrogenases in microorganisms, Thomas et al., 1993) or a Gly-XXX-Gly-X-Gly motif (short-chain alcohol dehydrogenases in animals, Jornvall et al., 1995). The identification of common structural domains through comparison of conserved sequences may provide the foundation necessary for subsequent structure-function analysis of the ASADH gene product domains. 187 Fig. 2. Southern blot analysis of the U. maydis ASADH gene. Genomic DNA from U. maydis strain 518 cells was digested with EcoRI (lanel), Psfl (Iane2), Smal (Iane3), and Xoal (Iane4), fractionated by electrophoresis in a 0.7% agarose gel, transferred to Zeta-Probe membrane, and hybridized with 32P-labeled 1.1 kb ASADH cDNA. Sizes of DNA markers are indicated in kb. 188 Fig. 3. PCR amplification and Southern blot analysis of ASADH gene from U. maydis genomic DNA and ASADH cDNA. Primers were derived from the 5' terminal (5'-ACGATGACGTCTTCTTCCTC-3') and 3' terminal (5'-CGAAAGGTAACCCTTGGCGA-3') regions of the U. maydis ASADH cDNA. PCR was carried out for 35 cycles of denaturation at 94 °C for 1 min, 1 min annealing at 58 °C, 1 min extension at 72 °C, followed by a final extension step for 10 min in the presence of 2.5 units of Taq-DNA polymerase. The amplified products were separated electrophoretically in a 1% agarose gel, visualized under UV following ethidium bromide staining (lane 1, 2, and 3), transferred to Zeta Probe membrane, and hybridized with 32P-labeled 1.1 kb ASADH cDNA (lane 4 and 5). Lane 1: 1kb DNA ladder, Lane 2: PCR product with genomic DNA as template, Lane 3: PCR product with ASADH cDNA as template, Lane 4: autoradiogram of the PCR product of lane 3, and Lane 5: autoradiogram of the PCR product of lane 4. 189 NADP-binding region Urn GLLGATGTVGQRFILQLAD 13 -31 Sc GVLGATGSVGQRFILLIAN 8 -26 L i AVLGATGSVGQRFIQLLDH 7 -25 Bs AWGATGAVGQQML KTLED 8 -26 Sm AIVGATGAVGTRMIQQLEQ 6 -24 Vc AlFGATGAVGETMLEVLQE 8 -26 Cg AWGATGQVGQVMRTLLEE 6 -23 Consensus GATG VG L Active site(C) and Core region IVTNANCSTTGIWPLKALEAKFG 151-174 IICISNCSTAGLVAPLKPLIEKFG 150-173 IITNSNCTIMGVTISLKPLLDRFG 142-165 IIANPNCSTIQMVAALEPIRKAYG 124-147 IIACPNCSTIQMMVALEPIRQKWG 122-145 IIANPNCSTIQMLVALKPIYDAVG 126-149 IIANPNCTTMAAMPVLKPLHDAAG 125-148 I NC L G Substrate-binding region Total No. of aa aa Identity Urn LKVSAHCNRVPVIDGHMECV 2 41-2 60 3 66 100% Sc IKVSAQCNRVAVSDGHTECI 241-260 365 53% L i FSISAHCNRVPVFDGHTVCV 226-245 349 43% Bs LQVAATCVRLPIQTGHSESV 235-254 346 21% Sm LPVSAHCVRVPILFSHSEAV 237-256 357 23% Vc IMVNPTCVRVPVFYGHAEAV 230-249 337 25% Cg LKVSGTCVRVPVFTGHTLTI 242-261 344 21% Consensus C R H Fig. 4. Conserved regions in ASADH-encoded amino acid sequences from Um (U. maydis), Bs (Bacillus subtilus), Cg (Corynebacterium glutamicum), Li {Leptospira interogans), Sm (Streptococcus mutans), Sc (Saccharomyces cerevisiae), and Vc (Vibrio cholerae). The amino acid sequence identity was obtained from the PALIGN program in the PC/GENE software. The Swiss-Prot database accession number for ASADH is Q04797 for Bs, P26511 for Cg, P41394 for Li, P10539 for Sm, P13663 for Sc, and P23247 for Vc. 190 Disruption of the U. maydis ASADH gene by insertion of a phleomycin resistant gene has been undertaken. Preliminary data show that disruption of the gene appears to be lethal to U. maydis (Kim S.H., Durrenberger F., Kronstad J., and Ellis B., unpublished data), implying that the ASADH gene is essential in this organism. 191 REFERENCES Cirillo, J.D., Barietta, R.G., Bloom, B.R. and Jocobs, W.R., Jr. (1991) A novel transposon trap for Mycobacteria: isolation and characterization of IS 1096. J. Bacteriol. 173:7772-7780. Gold, S.E. and Kronstad, J.W. (1994) Disruption of two genes for chitin synthase in the phytopathogenic fungus Ustilago maydis. Molecular Microbiology 11:987-902. Jdrnvall, H., Persson, B., Krook, M., Atrian, S., Gonzalez-Duarte, R., Jeffery, J. and Ghosh, D. (1995) Short-chain dehydrogenases/reductases (SDR). Biochemistry 34:6003-6013. Ouyang, J. and Viola, R.E. (1995) Use of structural comparison to select mutagenic targets in aspartate-p-semialdehyde dehydrogenase. Biochemistry 34:6394-6399. Pavia, N.L, Sun, Y., Dixon, R.A., Van Etten, H.D. and Hrazdina, G. (1994) Molecular cloning of isoflavone reductase from pea (Pisum sativum L): Evidence for a 3R-isoflavone intermediate in (±) pisatin biosynthesis. Arch. Biochem. Biophys. 312:501-510. Thomas, D., Barbey, R. and Surdin-Kerjan, Y. (1993) Evolutionary relationships between yeast and bacterial homoserine dehydrogenases. FEBS Lett. 323:289-293. 192 APPENDIX B A Partial cDNA Sequence Encoding the Ustilago maydis Peroxidase-Catalase During the studies on the cloning of the gene encoding PAL from U. maydis (chapter 3), a cDNA clone giving a positive signal during the immunoscreening a XZAPU U. maydis cDNA library was isolated and its nucleotide sequence was determined. The nucleotide sequence analysis of the 1725-bp E C O R I - X A J O I fragment insert of the cDNA clone revealed that the cDNA contains a 1593-bp open reading frame encoding a protein of 531 amino acids, a TAA stop codon, and a 132-bp 3'-untranslated region (Fig. 1). BLAST search through the Swiss-Prot database shows that the deduced 551 amino acid sequence has highest homology with the amino acid sequences of catalase-peroxidases from several organisms (Table 1). Based on this homology and the presence of the peroxidase proximal heme ligand (Fig. 1), this cDNA was tentatively designated as a partial cDNA encoding U. maydis peroxidase/catalase. Whether the gene product of this putative peroxidase/catalase gene is bifunctional, exhibiting a catalase and broad-spectrum peroxidase activities, remains to be determined. If it is bifunctional, this represents the first report on the partial structure of peroxidase/catalase-encoding gene from fungi. Cloning of the complete sequence of this gene would enable a more detailed study on the role of these proteins in Ustilago biology. 193 C G C A A T C T C G A C C A C G C T C T T G C C G C C T C G C A C A T G G G T C T T A T C T A C G T C A A C C C C G A G 60 R N L D H A L A A S H M G L I Y V N P E 20 G G A C C C A A C G G T G A G C C T G A C C C G G T T G N T G C C G C C C A C G A T A T C C G C A C C A C C T T C G G C 120 G P N G E P D P V X A A H D I R T T F G 40 R M A M N D E E | T V A L I A G G H T F | G 60 A A G A C T C A T G G T G C T G G T A A C C C A G A T C T C G T C G G C C C C G A A C C C A A C G G C G C T C C C A T C 2 4 0 K T H G A G N P D L V G P E P N G A P I 80 G A G G C T C A G G G C T T C G G T T G G A C C A G C A A G C A T G G T T C T G G T A A A G C T G G C G A T G C G A T T 300 E A Q G F G W T S K H G S G K A G D A I 100 A C C T C G G G T C T C G A G G T T G T C T G G A C T A G C A A G C C T A C C G A G T G G T C C A A C C T C T A C C T C 360 T S G L E V V W T S K P T E W S N L Y L 120 A A G T A C C T C T T T G A G T T C G A G T G G G A G C A C G A C A A G T C G C C C G C T G G C G C C A A C C A G T T T 420 K Y L F E F E W E H D K S P A G A N Q F 140 G T C G C C A A G A A T G C C G A C G C C A T C A T C C C C G A T C C C T T C G A C C C A T C C A A G A A G C G T C G T 480 V A K N A D A I I P D P F D P S K K R R 160 C C T A C T A T G C T C A C C A C C G A T C T A T C G T T G C G C T A C G A T C C T G C C T A C G A G A A G A T C T C G 540 P T M L T T D L S L R Y D P A Y E K I S 180 C G T C G C T T C C T T G A G A A C C A C G A C G A G T T T G C C G A C G C C T T T G C C C G T G C C T G G T T C A A A 600 R R F L E N H D E F A D A F A R A W F K 200 C T G C T C C A C C G T G A C A T G G G T C C T C G C G C C C G C T G G C T T G G A C C C G A G G T G C C C A A G G A G 660 L L H R D M G P R A R W L G P E V P K E 2 2 0 A T C C T T A T C T G G G A G G A C C C C G T G C C T A C C G C C G A T T A C G C T C T C G T G G A C G A C C G C G A C 720 I L I W E D P V P T A D Y A L V D D R D 2 4 0 C T T G C C G G C T T G A A G C A G G C T A T T T T T G C C A C T G G C G T C G A A C C T T C C A A G T T C C T T G C C 780 L A G L K Q A I F A T G V E P S K F L A 2 6 0 A C C G C C T G G G C T T C C G C T G C C A G C T A C C G A G A C A G T G A C A A G C G C G G C G G T G C C A A C G G T 840 T A W A S A A S Y R D S D K R G G A N G 2 8 0 G C T C G C A T C C G C C T T G C A C C G A T G A A G G A C T G G G A A G T C A A C A A T C C T C A G C A G C T C G C T 900 A R I R L A P M K D W E V N N P Q Q L A 300 G A G G T C A T C A A G G C T C T C G A G G G C G T T C A G C A G C A G T T C A A C T C T T C C A A C C A A G G T G G C 960 E V I K A L E G V Q Q Q F N S S N Q G G 320 A A G A A G A T T T C G A T T G C T G A C T T G A T C G T T C T C G C C G G T A A C G C A G C G C T T G A G A A G G C A 1020 K K I S I A D L I V L A G N A A L E K A 340 194 TCGGGTCTCCCCGTTCCCTTCACTCCTGGTCGTACTGATGCTACCCAGGAGCAGACCGAG 1080 S G L P V P F T P G R T D A T Q E Q T E 360 GTCGACACCTTCGAGTTCCTCAAGCCGGTCGCCGATGGCTTCCGCAATTACGGCCAGTCC 1140 V D T F E F L K P V A D G F R N Y G Q S 380 ACCGACCGTGTTTGCGCTGAACAGATCCTCATTGACCGCGCCAACCTGCTCACTCTCACC 1200 T D R V C A E Q I L I D R A N L L T L T 400 CCTCCCGAGCTCACTGTCCTCATCGGCGGTCTCCGCGCTCTTGGTCTCAACTACAACGGC 12 60 P P E L T V L I G G L R A L G L N Y N G 420 TCGTCACACGGTGTCTTGACTCACCGCCGAGGCCAGCTCTCGAACGACTTCTTTGTCAAC 1320 S S H G V L T H R R G Q L S N D F F V N 440 CTCCTCGACATGAGCACCGAGTGGAAGGCTGCTGACGGTGGCAAGGGCGAAGTCTTCGAC 1380 L L D M S T E W K A A D G G K G E V F D 460 GGTGTCGACCGCAAGTCAGGCCAGAAGAAGTGGTCTGCTACCCGTGCCGATCTTGTCTTT 1440 G V D R K S G Q K K W S A T R A D L V F 480 GGCTCTCAGGCTGAGCTTCGTGCCCTCGCCGAGAACTACGCTCAGGCCGACAACGCCGAC 1500 G S Q A E L R A L A E N Y A Q A D N A D 500 AAGTTCAAGAAGGACTTTGTGACTGCCTGGAACAAGGTTATGAACCTGGATCGTTTTGAC 1560 K F K K D F V T A W N K V M N L D R F D 520 GTCAAGAAGAGCAACATTGCCCGTGCCAGGTTCTAACCATGCTCCGCTCCAATCACTGAT 1620 V K K S N I A R A R F 531 GCTGTAGTAGTAGTAGCCATGATTTCCTTGCCCTATTCAGATTTATGGAATGTCTCTTTT 1680 ATGGAATCTTATTTTATTTTTTAACTTAAAAAAAAAAAAAAAAAA 1725 Fig. 1. Nucleotide sequence of a 1725-bp cDNA contained in a positively selected cDNA clone during the immunoscreening of U. maydis PAL cDNA clone (chapter 3). Deduced amino acid sequence of this cDNA is indicated below the respective codons. Numbers on the right margin indicates position relative to the C nucleotide or R amino acid in the beginning of sequences, respectively. Stop codon TAA is underlined. Predicted peroxidases proximal heme ligand signature (boxed), ATP/GTP-binding site motif A (P-loop) (letters in bold), and 2Fe-2S ferredoxin, iron-sulfur binding region signature (marked as •) are shown. 195 Table 1. BLAST search results with the protein sequence deduced from the U. maydis 1725-bp cDNA in Fig. 1. The results show the ten most similar protein sequences from the Swiss-Prot database. Rank Accession Organism Sequence Probability Number Description 1 P14412 Bacillus stearothermophilus Peroxidase/Catalase 2. 5e-218 2 Q04657 Mycobacterium intracellulars Peroxidase/Catalase 6. le- 216 3 P13029 Escherichia coli Catalase HPI (Hydroperoxidase I) 1. 3e-210 4 P46817 Mycobacterium vosis Peroxidase/Catalase 2. Oe-208 5 Q08129 Mycobacterium tuberculosis Peroxidase/Catalase 5. 2e-208 6 P17750 Salmonella typhimurium Catalase HPI (Hydroperoxidase I) 1. Oe- 024 7 P37743 Rhodobacter capsultus Peroxidase/Catalase 9. le- 05 8 P00431 Saccharomyces ceravisiae Cytochrome C Peroxidase Precursor 2. 9e-05 9 Q05431 Arabidopsis thaliana L-Ascorbate Peroxidase 3. 9e-05 10 P48534 Pisum sativum L-Ascorbate Peroxidase 0. 020 196 

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