Science, Faculty of
Microbiology and Immunology, Department of
DSpace
UBCV
Fortin, Pascal
Doctor of Philosophy - PhD
University of British Columbia
A multi-faceted approach was used to investigate and overcome the PCB-degrading limitations of the Bph enzymes, responsible for the catabolism of biphenyl. First, the reactivities of four evolutionarily divergent extradiol dioxygenases towards mono-, di- and trichlorinated 2,3-dihydroxybiphenyls (DHBs) were investigated: 2,3-dihydroxybiphenyl dioxygenase (EC 1.13.11.39) from Burkholderia xenovorans sp. LB400 (DHBD[sub LB400]), DHBD[sub P6]-I and DHBD[sub P6]-III from Rhodococcus globerulus P6 and 2,2',3-trihydroxybiphenyl dioxygenase from Sphingomonas sp. RW1 (THBD[sub RW1]). The specificity of each enzyme for particular DHBs differed by up to 3 orders of magnitude. Moreover, each enzyme cleaved at least one of the tested chlorinated DHBs better than DHB. However, no enzyme was able to cleave 2',6'-diCl DHB or 3,4-DHB, two recalcitrant PCB metabolites. In the second facet of the study, biological selections were designed to facilitate the engineering of PCB-degrading enzymes via directed evolution. Although these selections failed to link the desired activities to host cell viability, they showed promise for other biocatalysts. In addition, a broadly useful high-throughput colorimetric screen was developed. The latter was applied to increase the specificity of DoxG, an extradiol dioxygenase from Pseudomonas sp. CI8, for 3,4-DHB. A single round of directed evolution yielded DOXG[sub SMA2], a variant that cleaved 3,4-DHB ∼1000-fold more specifically than the wild-type enzyme. DOXG[sub SMA2] contained three substituted residues: L190M, S191W and L242S. The crystal structure of the DoxG:3,4-DHB binary complex indicates that residues at position 190 and 242 occur on opposite sides of the DHB-binding pocket and may interact directly with the distal ring of the substrate. Kinetic analyses revealed that the substitutions are anti-cooperative. Finally, characterization of BphK, the glutathione S-transferase from the bph pathway of B. xenovorans sp. LB400, revealed that the enzyme catalyzes the glutathione-dependant dehalogenation of certain inhibitory CI HOPDAs. BphK catalyzed the dechlorination of 3-Cl HOPDA with a specificity constant of ~10⁴ M⁻¹s⁻¹ in a reaction that utilized a ternary complex mechanism and 2 equivalents of GSH. The identified product of the reaction, HOPDA, is the substrate of the hydrolase from the bph pathway. The relatively low specificity constant of BphK for 3-Cl HOPDAs corroborate genetic evidence that the enzyme was recently recruited to the bph pathway to facilitate PCB degradation.
DEGRADATION OF POLYCHLORINATED BIPHENYL (PCB) METABOLITES: DIRECTED EVOLUTION AND E N Z Y M A T I C FORTUITY By Pascal Fortin B . S c , Universite Laval, 1999 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E OF D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E STUDIES ( M I C R O B I O L O G Y A N D I M M U N O L O G Y ) The University of British Columbia September 2005 © Pascal Fortin, 2005 ABSTRACT A multi-faceted approach was used to investigate and overcome the PCB-degrading limitations of the Bph enzymes, responsible for the catabolism of biphenyl. First, the reactivities of four evolutionarily divergent extradiol dioxygenases towards mono-, di- and trichlorinated 2,3-dihydroxybiphenyls (DHBs) were investigated: 2,3-dihydroxybiphenyl dioxygenase (EC 1.13.11.39) from Burkholderia xenovorans sp. LB400 (DHBDL B40o), D H B D P 6 - I and DHBD P 6 - I I I from Rhodococcus globerulus P6 and 2,2',3-trihydroxybiphenyl dioxygenase from Sphingomonas sp. RW1 ( T H B D R W i ) . The specificity of each enzyme for particular DHBs differed by up to 3 orders of magnitude. Moreover, each enzyme cleaved at least one of the tested chlorinated D H B s better than D H B . However, no enzyme was able to cleave 2' ,6 '-diCl D H B or 3,4-DHB, two recalcitrant PCB metabolites. In the second facet of the study, biological selections were designed to facilitate the engineering of PCB-degrading enzymes via directed evolution. Although these selections failed to link the desired activities to host cell viability, they showed promise for other biocatalysts. In addition, a broadly useful high-throughput colorimetric screen was developed. The latter was applied to increase the specificity of DoxG, an extradiol dioxygenase from Pseudomonas sp. CI8 , for 3,4-DHB. A single round of directed evolution yielded DOXGSMA2, a variant that cleaved 3,4-DHB -1000-fold more specifically than the wild-type enzyme. DOXGSMA2 contained three substituted residues: L190M, S191W and L242S. The crystal structure of the DoxG:3,4-D H B binary complex indicates that residues at position 190 and 242 occur on opposite sides of the DHB-binding pocket and may interact directly with the distal ring of the substrate. Kinetic analyses revealed that the substitutions are anti-cooperative. Finally, characterization of BphK, the glutathione S-transferase from the bph pathway of B. xenovorans sp. LB400, revealed that the enzyme catalyzes the glutathione-dependant dehalogenation of certain inhibitory CI HOPDAs. BphK catalyzed the dechlorination of 3-C1 H O P D A with a specificity constant of ~10 4 M"'s _ 1 in a reaction that utilized a ternary complex mechanism and 2 equivalents of G S H . The identified product of the reaction, H O P D A , is the substrate of the hydrolase from the bph pathway. The relatively low specificity constant of BphK for 3-C1 H O P D A s corroborate genetic evidence that the enzyme was recently recruited to the bph pathway to facilitate P C B degradation. T A B L E OF CONTENTS ABSTRACT ii T A B L E OF CONTENTS iv LIST OF TABLES vii LIST OF FIGURES viii ABBREVIATIONS x F O R E W O R D xii A C K N O W L E D G M E N T xiii DEDICATION xv 1. INTRODUCTION 1 1.1 Aromatic compounds in Nature 1 1.2 Polychlorinated biphenyls (PCBs) 1 1.3 Catabolism of aromatic compounds 2 1.4 The bph pathway 5 1.5 Extradiol dioxygenases 7 1.6 Bacterial GSTs 11 1.7 Metabolic Blocks 13 1.8 Unblocking the pathway 16 1.9 Enzyme engineering by directed evolution 18 1.10 Exploring the sequence space: mutagenesis techniques and library creation 18 1.11 Biological selections and screens 21 1.12 Aim of this study 23 2. MATERIALS AND METHODS 24 2.1 Chemicals 24 2.2 Oligonucleotides and D N A sequencing 24 2.3 Bacterial strains, plasmids and growth conditions 25 2.3.1 Transformation and conjugation 28 2.3.2 Cloning of the dbfB gene 28 2.3.3 Cloning of the bphK gene 29 2.4 Construction of strains for Biological Selections and Screens (SHC systems) 29 2.4.1 Cloning the reporter genes 29 2.4.1.1 Cloning of the chloramphenicol acetyl-transferase {cat) gene 30 2.4.1.2 Cloning of the tetracycline resistance (tetC) gene 30 2.4.1.3 Cloning of the lux cassette 30 2.4.2 Construction of SHC 1 and SHC2 30 2.4.3 Construction of SHC4, SHC7, SHC11 and SHC 12 32 2.4.4 Cloning dbfB and bphD in p M A D and pUCP24 32 2.5 Expression tests of the reporter genes in the SHC systems 33 2.6 Directed evolution of doxG: mutagenesis procedures 33 2.6.1 Error-prone P C R 34 2.6.2 Saturation mutagenesis 34 2.6.3 D N A shuffling 34 2.6.4 DoxG L242S and L190M/S191W variants 36 2.7 Cloning conditions and library construction 36 2.7.1 Determination of the required library size 36 2.8 Colorimetric screen 37 2.9 Activity assays in 96 wells microplates 37 2.10 Protein purification 39 2.10.1 Purification of THBDRWI 39 2.10.2 Purification of DOXG SMA2 41 2.10.3 Purification of BphK 41 2.11 Handling and characterization of dioxygenase samples 42 2.11.1 Extradiol dioxygenases steady-state kinetic experiments 42 2.11.2 Spectrophotometric assay of ring-cleavage reaction 43 2.11.3 Determination of partition ratio and inactivation constants 43 2.11.4 H P L C analysis of cleavage products 44 2.12 Handling and characterization of BphK samples 45 2.12.1 Ability of BphK to transform PCB metabolites 45 2.12.2 Evaluation of BphK steady-state kinetic parameters 46 2.12.3 Characterization of the BphK-catalyzed transformation product of 3-C1 H O P D A 46 2.13 Protein structure modeling of THBDRWI 47 3. RESULTS 48 3.1 Transformation of P C B metabolites by evolutionarily diverse extradiol diooxygenases 48 3.1.1 Purification of THBDRWI and other D H B D s 48 3.1.2 Physical properties of HOPDAs 49 3.1.3 Reactivity of D H B D L B 4 0 0 , D H B D P 6 - I , D H B D P 6 - I I I and THBDRWI with chlorinated D H B 50 3.2 Biological selection and screening host cells (SHCs) 54 3.2.1 E. coli SHC1: towards a benzoate-inducible chloramphenicol resistance system 55 3.2.2 SHC2: a bimodular benzoate-inducible chloramphenicol resistance system 57 3.2.3 E. coli SHC4 and P. putida SHC7: bimodular benzoate-inducible tetracycline resistance 59 3.2.4 P. putida SHC11 and E. coli SHC 12: luminescence-based biological screening host cells 60 3.2.5 Colony screen 63 3.3 Directed evolution of DoxG 64 3.3.1 Library generation and analysis 65 3.3.1.1 Error-prone PCR 65 3.3.1.2 Saturation mutagenesis 66 3.3.1.3 D N A shuffling 68 3.3.2 Characterization of WT and variant DoxG 70 3.3.2.1 Purification of DoxG and DOXGSMA2 70 3.3.2.2 Steady-state kinetic analysis 70 3.3.2.3 Inverse analysis of DOXGSMA2 mutations 71 3.3.2.4 H P L C analysis of 3,4-DHB cleavage products 72 3.4 Transformation of P C B metabolites by BphK 74 3.4.1 Expression and Purification of BphK 74 3.4.2 BphK-catalyzed transformation of chlorinated DHBs, H O P D A s and 4 -CBA 75 3.4.3 Demonstration that BphK catalyzes the dechlorination of 3-C1 HOPDAs 75 3.4.4 Steady-state kinetics with 3-C1 H O P D A , 3,9,11-triCl H O P D A and G S H 77 3.4.4.1 Steady state kinetics using fixed concentrations of G S H and HOPDAs..77 3.4.4.2 Steady state kinetics using varied concentrations of G S H and H O P D A s 77 4. DISCUSSION 82 4.1 Reactivity of D H B D s 82 4.2 Selection and Screen development 86 4.3 Directed evolution of DoxG 94 4.4 Transformation of P C B metabolites by BphK 96 4.5 Concluding remarks 103 5. BIBLIOGRAPHY 105 LIST OF TABLES Table 1. Oligonucleotides used in this study 25 Table 2. Bacterial strains used in this study 26 Table 3. Plasmids used in this study 27 Table 4. Purification details of THBDRWI 48 Table 5. Molar extinction coefficients of HOPDAs 49 Table 6. Apparent steady-state kinetic parameters and inactivation parameters of DHBDLBWO, D H B D P 6 - I , DHBDp 6-III and THBDRWI for chlorinated DHBs 53 Table 7. Regulation of chloramphenicol resistance by benzoate in E. coli S17.lA.pir ( p C N B l C A T ) 56 Table 8. E. coli SHC2 ( p M A D D B ) growth in the presence of chloramphenicol, benzoate and D H B 58 Table 9. Growth of SHC7 in the presence of, benzoate and D H B 60 Table 10. Relative activities of selected DoxG variants obtained by error-prone P C R 66 Table 11. Relative activities of selected DoxG variants obtained saturation mutagenesis 68 Table 12. Purification of DOXGSMA2 70 Table 13. Apparent steady-state kinetic parameters of DoxG and variant S M A 2 71 Table 14. Apparent relative activity of DoxG variants against 1,2-DHN, D H B and 3,4-DHB...72 Table 15. Purification details of BphK expressed in E. coli GJ1158 74 Table 16. Steady-state kinetic parameters of BphK for chlorinated H O P D A s 79 LIST OF FIGURES Figure 1. Degradation pathways of polyaromatic compounds and central intermediates 3 Figure 2. The bph pathway responsible for the degradation of biphenyl 6 Figure 3. Domain structure of DoxG from Pseudomonas sp. strain CI8 7 Figure 4. Structure of the active site of DHBDLB4OO 9 Figure 5. Proposed catalytic mechanism of extradiol dioxygenases 10 Figure 6. Metabolic blocks of the upper bph pathway 14 Figure 7. Reactions catalyzed by DoxG 17 Figure 8. General directed evolution strategy 19 Figure 9. Organization of the mini-transposons used to construct the SHC strains 31 Figure 10. Imaging strategy used to facilitate colony screening 38 Figure 11. Molecular numbering of D H B and corresponding H O P D A 49 Figure 12. Steady-state cleavage of D H B by T H B D R W i 52 Figure 13. Principles used to design the selection-based SHCs for the directed evolution of PCB-degrading enzymes 54 Figure 14. The two mini-Tn5 modules that were stably inserted into the chromosome of E. coli CC118Rif to create E. coli SHC2 57 Figure 15. Induction of luminescence by benzoate in P. putida SHC11 and E. coli SHC 12 61 Figure 16. Luminescence of SHC 12 (pMADBD) in the presence of D H B 62 Figure 17. Quantification of ring-cleavage activity in plated colonies using digital imaging.... 64 Figure 18. The structure of DoxG bound to 3,4-DHB 67 Figure 19. The structure of DoxG bound to 3,4-DHB showing the S M A 2 variant mutations... 69 Figure 20. Inverse analysis of the effect of S M A 2 substitutions on DoxG activity 73 Figure 21. Coomassie Blue-stained SDS-polyacrylamide gels of purified BphK 74 Figure 22. Dechlorination of 3-C1 H O P D A by BphK 76 Figure 23. U V - V i s absorption spectra of 3-C1 H O P D A , H O P D A and product of BphK-catalyzed transformation of 3-C1 H O P D A 76 Figure 24. Steady-state transformation of 3-C1 H O P D A by BphK 78 Figure 25. Steady-state analyses of the BphK-catalyzed transformation of 3-C1 HOPDAs . The BphK-catalyzed transformation of 3-C1 H O P D A 80 Figure 26. Primary plots of steady-state the BphK-catalyzed transformation of 3-C1 and 3,9,11-triCl HOPDAs 81 Figure 27. Stereo view of the structure of the active site of DHBDLB400 bound to D H B and the modeled active site of THBDRWI bound to 2 ' ,6 '-diCl D H B 85 Figure 28. Dechlorination of 3-C1 H O P D A by BphK 97 Figure 29. The proposed mechanism for the T C H Q dehalogenase-catalyzed dechlorination of T C H Q and a possible mechanism for the BphK-catalyzed dehalogenation of 3-C1 HOPDA....101 ABBREVIATIONS 1,2-DHN 1,2-dihydroxynaphthalene 3 , 4 - D H B 3,4-dihy droxybipheny 1 3CC 3-C1 catechol 4 - C B A 4-chlorobenzoate 4-Phenyl H O D A 2-hydroxy-4-phenyl-6-oxo-hexa-2,4-dienoic acid 5-phenyl H O D A 2-hydroxy-5-phenyl-6-oxo-hexa-2,4-dienoic acid B P D O biphenyl dioxygenase (EC 1.14.12.18) C230 catechol 2,3-dioxygenase (EC 1.13.11.2) C D B N 1 -chloro-2,4-dinitrobenzene D C M dichloromethane D H B 2,3-dihydroxybiphenyl D H B D 2,3-dihydroxybiphenyl 1,2-dioxygenase (EC 1.13.11.39) DHBDLB400 D H B D from Burkholderia xenovorans LB400 D H B D P 6 - I D H B D - I from Rhodococcus globerulus P6 DHBDp 6-III DHBD-III from Rhodococcus globerulus P6 DTT dithiotreitol F A C S fluorescence activated cell sorting GFP green fluorescent protein GS" thiolate form of glutathione GSSG glutathione disulfide G S H glutathione GST glutathione S-transferase (2.5.1.18) H G O homogentisate dioxygenase (EC 1.13.11.5) H O P D A 2-hydroxy-6-oxo-6-phenyl-2,4-dienoate IPTG isopropyl-/?-D-thiogalactopyranoside I T C H Y Iterative Truncation for the Creation of HYbr id enzymes M A A maleylacetoaacetate M A A I maleylacetoaacetate isomerase (EC 5.2.1.2) M P maleylpyruvate MPI maleylpyruvate isomerase (EC 5.2.1.2) PAHs polycyclic aromatic hydrocarbons PCBs polychlorinated biphenyls PCP pentachlorophenol R A C H I T T Random chimeragenesis on transient templates R L U relative light units SDS P A G E Sodium dodecyl sulphate polyacrylamide gel ROS reactive oxygen species SHC selection and screening host cell SNAr nucleophilic aromatic substitution StEP staggered extension process T C H Q tetrachlorohydroquinone THBDRWI 2,2',3-trihydroxybiphenyl dioxygenase from Sphingomonas sp. RW1 (EC 1.13.11.-) tR retention time triCHQ trichlorohydroquinone triCBQ trichlorobenzoquinone V O C vicinal oxygen chelate WT wild-type FOREWORD Parts of this work have been published in refereed journals. Manuscripts concerning other parts of this thesis are presently in preparation and wil l be submitted shortly for publication. The characterization of the specificity of four evolutionarily divergent extradiol dioxygenases which resulted in the identification of enzymes with higher specificities for some chlorinated metabolites and also led to the conclusion that all extradiol dioxygenases studied so far are affected by the critical P C B metabolites 2 ' ,6 ' -diCl DHBs . This study was published in Journal of Bacteriology (Fortin P.D., Lo A.T.F . , Haro M.A.,Kaschabek S.R., Reineke W., Eltis L .D . 2004. Evolutionarily Divergent Extradiol Dioxygenases Possess Higher Specificities for PCB Metabolites. J. Bacteriol. 187, 415-421). The manuscript concerning the directed evolution of DoxG has been written and wil l be submitted shortly. It wi l l include the structure of the variants DOXGSMA2, which is presently under completion. The preliminary characterization of BphK, which led to the identification of a dehalogenase activity against critical P C B metabolites, wi l l be part of a manuscript that wil l be submitted shortly. A C K N O W L E D G M E N T I would like to thank my supervisor Lindsay Eltis for his guidance, scientific criticism, great patience and support. I would like to thank all members of the Eltis Lab, past and present, who make/made the lab a livable place. Special thanks go to Frederic Vaillancourt for his great mentorship in the early years of my studies. I would also like to acknowledge Geoff Horsman, Carly Huitema and Leticia Gomez G i l for hepful discussions and feedback and Cheryl Whiting for valuable technical assistance. Many thanks to Thomas Heuser and Sachi Okamoto for contributing to my mental health. Special thanks must go to the undergraduate students who worked with me, some on parts of the present PhD work. I would like to acknowledge Matt Witt, who was of great assistance with the construction, often frustrating, of the first biological selection systems. Many thanks go to Andy Lo and Marvin Hsiao for their work on the characterization of THBDRWI , and Ambrose Wong, who was involved with the cloning of bphK. I would like to thank Mark Wang for his valuable work of on the BphK project. I would finally like to acknowledge the great work of Manpreet Cheema and Gina Lee, who were involved in projects not described in the present thesis. I would also like to acknowledge the great advice, support and friendship of Daryl Smith. Special thanks to Iain MacPherson for his help and ideas, friendship and surfing expeditions. Many thanks to my advisory committee members, B i l l Mohn, Michael Murphy and Stephen Withers for their guidance and for reviewing my thesis. Many Thanks also to David Neau and Jeffrey T. Bolin for providing me with great crystal structures of DoxG and to Victor Snieckus and his group for synthesizing and providing the highly pure substrates used in this study. I would like to acknowledge the Natural Sciences and Engineering Research Council of Canada and the Fonds pour la formation des Chercheurs et l 'Aide a la Recherche (FCAR) for postgraduate scholarships throughout my PhD studies. I would also like acknowledge the Gerard D. Levesque (GDL) Foundation to honor me with the postgraduate G D L award. J'aimerais remercier ma famille pour leur grand support et comprehension tout au cours de mes etudes doctorales. Meme si mes recherches m'ont mene a plusieurs milliers de kilometres de la maison, vous etes toujours rester pres de moi. J'aimerais aussi remercier mes grands amis Sebastien, Catherine et Danick. Nos carrieres nous ont amenes au quatre coins du pays mais nous avons su garder contact. J'apprecie grandement votre amitie et vos encouragements aux cours de mes longues annees d'etudes graduees. Finally, I would like to thank my best friend, my wife Grace, for her great support, patience and comprehension. You have encouraged me, inspired me and supported my career plans all along. Thank you. A la memoire de mon Pere \ 1. Introduction 1.1 Aromatic compounds in Nature. The benzene ring is the second most widely distributed chemical structure in Nature after the glucosyl group: it constitutes 25% of the earth's biomass, and is particularly prevalent within the biopolymer lignin (Gibson and S. Harwood 2002; Diaz 2004). Aromatic compounds such as benzene are fully conjugated molecules. The derealization of electrons in the n orbitals of these compounds increases their physicochemical stability through resonance energy. Man has sought to exploit this stability in developing chemicals for various applications. The discovery that chlorination often further increased this stability, together with the development in the 1950s of new organic chemistry techniques led to the synthesis of new classes of compounds. A s a result, the past century was marked by a rapidly increasing release of man-made (xenobiotic) aromatic compounds in the environment. Examples of such xenobiotics include dioxins, polycyclic aromatic hydrocarbons (PAHs), poly chlorinated biphenyls (PCBs) and halogenated aromatic pesticides such as D D T . Unlike the naturally occurring aromatic compounds, xenobiotics tend to be recalcitrant to biodegradation and accumulate in the environment (Oberg 2002). 1.2 Polychlorinated biphenyls (PCBs) One of the most widely distributed classes of chlorinated pollutants in the environment is that of PCBs. A n estimated 2 billion kilograms of these compounds were produced during the 20 t h century (World Health Organization 2003) for use in numerous industrial applications, including as plasticizers, flame retardants and dielectrics. PCBs were manufactured as complex mixtures of 209 possible "congeners", which differ in the number and position of chloro substituents on the biphenyl rings. It is the 100 or so congeners that contain 5 to 7 chlorines that occur in many commercial formulations that are prevalent in environmental matrices, and are the most bio-accumulating. Although the production and widespread usage of PCBs was banned in the industrial world in the 1970s, their destruction is of continued relevance due to their pervasiveness and persistence, their impact on fragile ecosystems and human health concerns. The half-life of PCBs in the human blood stream ranges from 1 to 10 years depending on the congener (Phillips et al. 1989). P C B exposures have been linked to a variety of health problems including cancer, neurological complications, dermatologic abnormalities and compromised reproductive functions (Masuda 2003; Seegal 2003). 1.3 Catabolism of aromatic compounds Microorganisms are the only living creatures that grow on aromatic compounds as sole source of carbon and energy. As such, these microbial catabolic activities play a critical role in the global carbon cycle. Microorganisms have evolved strategies to catabolize aromatic compounds in the absence and the presence of oxygen. In both cases, the aromatic ring is activated prior to its cleavage and transformation to central metabolites. Under anaerobic conditions, the compounds are transformed to one of three intermediates (benzoyl-CoA, resorcinol and phloroglucinol) which are then dearomatized and hydrolytically ring-cleaved (Heider and Fuchs 1997; Schink et al. 2000). The resulting aliphatic acids are further catabolized via p-decarboxylation. By contrast, in aerobic catabolism, the aromatic ring is usually activated using molecular oxygen to yield a dihydroxylated intermediate, such as a catechol, protocatechuate or gentisate (Dagley 1978; Dagley 1986; Harayama and Timmis 1992). These intermediates are then cleaved oxygenolytically by a ring-cleavage dioxygenase that incorporates both atoms of molecular oxygen into the ring-opened product (Figure 1). There biphenyl naphthalene dibenzofuran dibenzo-p-dioxin I 3 I 3 I 3 I 3 1 1 1 I gentisate pathway Figure 1. Degradation pathways of polyaromatic compounds and central intermediates. Reaction labeled with a are catalyzed by ring-hydroxylating dioxygenase. Reactions labeled with b are catalyzed by ring-cleavage dioxygenase. Central intermediates protocatechuic acid and catechol can be cleaved by an intradiol dioxygenase (ortho) or an extradiol dioxygenase (meta) (Inspired from Vaillancourt 2002). are two classes of ring cleavage dioxygenases that differ in their structures and mechanisms: intradiol dioxygenases and extradiol dioxygenases (Harayama and Rekik 1989). Intradiol dioxygenases cleave the bond situated between the two hydroxyl groups and typically depend on mononuclear non-heme Fe (Vetting et al. 2000). In contrast, extradiol dioxygenases cleave a bond adjacent to the two hydroxyl groups and typically depend on mononuclear non-heme Fe (Tiirola et al. 2002), although some enzymes use M n (Que and Reynolds 2000). Finally, a new principle for the aerobic metabolism of aromatic compounds was recently described in Azoarcus evansii (Zaar et al. 2004). In this pathway, which is also found in Burkholderia xenovorans LB400 (Denef et al. 2004), compounds such as benzoate are first transformed to benzoyl-CoA before being dihydroxylated. The ring is then cleaved hydrolytically. Aerobic pathways for the catabolism of aromatic compounds are widely distributed in both Gram negative and Gram positive soil bacteria. These pathways have been particularly well characterized in Pseudomonads and actinomycetes, such as Rhodococcus (Harayama and Timmis 1989; Masai et al. 1995). A large number of xenobiotics resemble naturally occurring aromatic compounds. Thus, it is not surprising that many of the former are at least partially transformed by existing bacterial catabolic pathways. The transformation of recently generated xenobiotics by bacterial pathways has two interesting aspects. First, it has fueled the development of technologies that harness microbial catabolic capabilities to destroy environmental pollutants. For example, PCBs can be destroyed in contaminated soils through sequential treatment with anaerobic and aerobic bacterial strains (Maltseva et al. 1999). Second, it provides an opportunity to study the evolution of metabolic pathways. Thus, several lines of evidence indicate that the pathway used by Sphingobium chlorophenolicum to aerobically degrade pentachlorophenol (PCP) has only recently evolved through the patchwork assembly of two other catabolic pathways and continues to evolve (Copley 2000; Tiirola et al. 2002). In fact, pentachlorophenol hydroxylase and 2,6-dichlorohydroquinone dioxygenase, the first and third enzymes of the PCP degradation pathway, may have been recruited from a pathway involved in degradation of a naturally occurring chlorinated phenol. On the other hand, the reductive dehalogenase catalyzing the second step of the pathway may have originated from a maleylacetoacetate isomerase normally involved in degradation of tyrosine (Anandarajah et al. 2000). 1.4 The bph pathway A wide variety of microorganisms are able to aerobically transform some of the 209 possible P C B congeners via the bph pathway, which degrades biphenyl to benzoate and 2-hydroxypenta-2,4-dienoate (Abramowicz 1990; Furukawa 2000). Much effort has been invested in studying the bph pathway to improve our understanding of its enzymatic make-up, to identify the steps that limit P C B transformation, and to improve its PCB-degrading capabilities. The bph pathway can be divided into the upper and lower pathways (Figure 2B). The upper bph pathway consists of four catabolic steps that transform biphenyl to benzoate and 2-hydroxypenta-2,4-dienoate. The first step is catalyzed by biphenyl dioxygenase (BPDO), a typical ring-hydroxylating dioxygenase comprising three components: an FAD-containing reductase (encoded by bphG), a Rieske-type ferredoxin (bphF) and a two-subunit terminal oxygenase (bphAE). The latter contains a Rieske cluster and a mononuclear iron centre that is part of the active site (Haddock and Gibson 1995; Hurtubise et al. 1995; Butler and Mason 1997). B P D O catalyzes the addition of O2 and two NADH-derived reducing equivalents to biphenyl, yielding cw-2,3-dihydro-2,3-dihydroxybiphenyl. The second step of the upper bph pathway is catalyzed by BphB, an enzyme that carries out the NAD +-dependent dehydrogenation of c/5-2,3-dihydro-2,3-dihydroxybiphenyl to form 2,3-dihydroxybiphenyl (DHB), a catechol (Seeger et al. 1995b; Barriault et al. 1999; Bruhlmann and Chen 1999). The Z>/?/zC-encoded 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHBD; E C 1.13.11.39) is the third enzyme of the bph pathway. D H B D catalyzes the extradiol cleavage of D H B , incorporating O2 into the substrate and yielding 2-hydroxy-6-oxo-6-phenyl-2,4-dienoate (Harayama and Timmis 1992). The last step of the upper bph pathway is catalyzed by BphD, or H O P D A hydrolase. This enzyme hydrolyses H O P D A at a carbon-carbon bond to yield benzoate and 2-hydroxypenta-2,4-dienoate (Omori et al. 1986). In some bacteria, including B. xenovorans LB400 , a glutathione S-transferase (GST; encoded by bphK) is associated with the upper bph pathway. This enzyme is not essential for growth on biphenyl and its physiological role is unclear (Bartels et al. 1999). The two products of the upper bph pathway are catabolized by separate pathways. 2-Hydroxypenta-2,4-dienoate is transformed to Krebs cycle intermediates by the three enzymes of the lower bph pathway: 2-hydroxypenta-2,4-dienoate hydratase (BphH), an acetaldehyde dehydrogenase (acylating; BphJ) and 4-hydroxy-2-oxovalerate aldolase (BphI) (Focht and Reineke 2002). Benzoate can be catabolized either via the dihydroxylation and oxygenolytic ring bphA^)|bphE^)| orfl ^>[bphF^>|bphG^>|bphB^>|bphC^>[bphK^>[bphH^>[bphJ^)|bphl ^>|bphD^> B || BphAEFG (BPDO) 0 2 ' NADH HO^COOH OH BphC OH (DHBD) 0, 2,3-dihydroxybiphenyl BphD biphenyl 2,3-dihydro-2,3-dihydroxybiphenyl 2-hydroxy-6-oxo-6-pheny lhexa-2,4-d ienoic COOH benzoic acid To benzoate degradation pathway HO^COOH T 2-hydroxypenta-2,4-dienoic acid H2oj BphH CoA CoASH J acetyl-CoA pyruvate acetaldehyde 4-hydroxy-2-oxovaleric acid Figure 2. The bph pathway responsible for the degradation of biphenyl. A ) Organization of the bph operon in B. xenovorans LB400. Open reading frames and genes of unknown function are represented by white arrows. B) bph metabolic pathway. Figure 3. Domain structure of D o x G from Pseudomonas sp. strain C I 8. The N-and C-terminal domains are shown in blue and red, respectively. Two different shades are used to show the (3aP(3(3 modules. The ferrous iron of the active site is represented as a green sphere. Figure made using P y M O L (Delano 2005). cleavage or via the benzoyl-CoA pathway. Interestingly, LB400 contains both pathways, and appears to utilize them simultaneously under some conditions (Denef et al. 2005). 1.5 Extradiol dioxygenases Extradiol dioxygenases depend on a mononuclear divalent metal ion, usually F e 2 + , for their enzymatic activity. These enzymes belong to one of three evolutionarily divergent superfamilies (Eltis and Bo l in 1996; Dunwell et al. 2001) although their mechanisms and metal ion environments are conserved, as discussed below. Type I extradiol dioxygenases belong to the vicinal oxygen chelate ( V O C ) superfamily (Armstrong 2000). The V O C structural fold is characterized by two copies of a module comprising 4 P-strands and 1 a-helix in the order pappp. A s seen in the structure of D o x G , the 1,2-dihydroxynaphthalene dioxygenase from Pseudomonas sp. strain C I 8 (Figure 3), the two Pappp modules form a funnel with the F e 2 + -containing active site inside (Neau 2004). Type I enzymes can contain either one copy of these domains, as exemplified by DHBDp6-III from Rhodococcus globerulus P6, or two copies of the domain, as exemplified by DHBDLB4OO fr°m B. xenovorans LB400, DHBDp6-I from R. globerulus P6 and 2,2',3-trihydroxybiphenyl dioxygenase from Sphingomonas sp. RW1 (THBDRWI) . Considerable variability in quaternary structure exists. For example, DoxG is octameric (Neau 2004) whereas THBDRWI is monomeric (Happe et al. 1993). In two-domain enzymes, only the C-terminal domain harbours an active site. Phylogenetic analyses suggest that the evolution of type I extradiol dioxygenases involved two duplication events followed by the divergence of one- and two-domain enzymes (Eltis and Bolin 1996). Type II extradiol dioxygenases are made up of either one or two subunits and appear to possess a unique fold (Sugimoto et al. 1999). The crystal structure of protocatechuate 4,5-dioxygenase, a two-subunit enzyme, revealed that the small subunit comprises 10 a-helices and that the large subunit consisted of an a/p scaffold composed of 11 P strands, 9 a-helices and one 3 io helix (Sugimoto et al. 1999). The one-subunit enzymes are homologous to the large subunit of the two-subunit enzymes (Spence et al. 1996). Type III extradiol dioxygenases belong to the cupin superfamily (Dunwell et al. 2001). These enzymes include homogentisate dioxygenase (HGO), which is involved in the catabolism of phenylalanine and tyrosine in all organisms, including Man. The proteins of the cupin superfamily contain at least one domain made up of 6 antiparallel P-sheets that form a P-barrel structure. H G O is composed of two copies of this domain, the C-terminal of which forms the Fe -containing active site. The active site divalent metal ion in all extradiol dioxygenases is ligated to the polypeptide by 2 histidines and a glutamate in what has been termed as a 2-his-l-carboxylate facial triad. In DHBDLB400, these ligands are His l46 , His210 and Glu260 (Figure 4A). Three other active site residues, His l95 , His241 and Tyr250, are conserved among Type I extradiol Figure 4. Structure of the active site of DHBD L B400 without substrate (A) and bound to D H B (B). Carbon atoms are shown in straw (protein) and yellow (DHB) . Oxygen, nitrogen, and iron atoms are coloured red, blue and green, respectively. The iron ligands are His 146, His210 and Glu260. Conserved residues thought to play a catalytic role are His 195, His241 and Tyr250. Structure made using P D B files 1 H A N (A) and 1 K M Y (B) (Han et al. 1995). dioxygenases and appear to be playing a key role in the catalytic mechanism. Equivalent residues are also found in type II and III extradiol dioxygenases. Spectroscopic, biochemical and structural studies support a compulsory-order mechanism in which the catechol first binds to the active site Fe as a monoanion in a bidentate manner (Han et al. 1995; Vaillancourt et al. 2002a). Substrate deprotonation is likely assisted by His241. In the proposed mechanism, subsequent O2 binding to the Fe, followed by the iron-mediated electron transfer from the catechol to O2, yields a semiquinone-Fe -superoxide intermediate which is thought to be stabilized by His 195. This species then reacts to give a C-2 iron-alkylperoxo intermediate, which undergoes alkenyl migration and 0 - 0 bond cleavage to give an unsaturated lactone intermediate and an Fe -bound hydroxide anion. The latter hydrolyzes the lactone to yield the reaction product (Shu et al. 1995; Bugg and L i n 2001). Figure 5 illustrates the proposed catalytic mechanism. apoenzyme Figure 5. Proposed catalytic mechanism of extradiol dioxygenases. The active site iron of the resting enzyme, which begins and ends the catalytic cycle, is depicted in the upper left hand corner with its 5 ligands. For clarity, the displacement of solvent species from the ferrous center is not explicitly shown. Two potential routes for inactivation are also shown. (Inspired from Vaillancourt 2002). Extradiol dioxygenases are susceptible to mechanism-based inactivation (Mitchell et al. 1963; Klecka and Gibson 1981). Biochemical studies indicate that this inactivation is due to the oxidation of the active site iron to F e 3 + via the dissociation of superoxide from the A E A Scheme 1: E +• A.+ O , -*==*• E A + 0 2 « i = * E A 0 2 E + P + ° 2 | i ' l | A E* E* note: E, enzyme; A, catecholic substrate; E*. inactivated enzyme; P, product enzyme:catechol:dioxygen ternary complex (Vaillancourt et al. 2002b). The active site iron can also be oxidized in the absence of catechol, but at a much lower rate (rate constant j\ \ scheme 1). Mechanism-based inactivation may be characterized by j$, the rate constant of inactivation, and the partition coefficient, the ratio of kcat to 73. The modes of inhibition of extradiol dioxygenases during steady-state turnover are summarized in Scheme 1. Substrates with low specificity often inactivate the enzyme more readily and are characterized by higher/3's and lower partition ratios. One particular potent suicide substrate is 3-C1 catechol (3CC), which can arise from the transformation of 3-C1 biphenyls. The partition coefficient of DHBD L B 400 for 3CC is 8, which is 104-fold lower than for D H B (Vaillancourt et al. 1998; Vaillancourt et al. 2002b). Similarly, the partition coefficient for 3CC of catechol 2,3-dioxygenase (C230) from the T O L pathway of Pseudomonas putida mt-2 is 1,400-fold lower than for its natural substrate, catechol (Wasserfallen et al. 1991; Cerdan et al. 1994). In some pathways, a 2Fe-2S ferredoxin functions to maintain the extradiol dioxygenase in the active state by reducing any oxidized enzyme (Polissi and Harayama 1993; Hugo et al. 2000; Tropel et al. 2002). Such a ferredoxin is present in the T O L pathway but absent from the bph pathway. 1.6 Bacterial GSTs Glutathione transferases (GSTs) constitute one of nature's most versatile enzymes, catalyzing a wide range of biochemical reactions involving the conjugation of glutathione (GSH) to electrophilic compounds (Armstrong 1997; Sheehan et al. 2001). Their substrates include natural and xenobiotic compounds, and their activities are often linked to cellular detoxification. They are believed to have evolved from an ancient thioredoxin enzyme superfamily to protect the cell against the rising oxidative stress generated by the advent of photosynthesis. Accordingly, it was recently proposed that Ochrobactrum anthropi induced the expression of a GST during the increased release of reactive oxygen species (ROS) arising from the aerobic degradation of phenol and 4-chlorophenol (Tamburro et al. 2004). In general, eukaryotic GSTs are much better characterized than their prokaryotic counterparts (Vuilleumier 1997), although recent genome sequencing efforts have revealed that some bacteria harbour many GSTs. For example, several pseudomonad strains possess over 10 genes predicted to encode GSTs (Vuilleumier and Pagni 2002). Many bacterial GSTs occur in pathways responsible for the aerobic catabolism of aromatic compounds. Some of these GSTs catalyze dehalogenation or carbon-carbon double bond isomerization. For example, Methylobacterium dichloromethanicum D M 4 contains a GSH-dependent dehalogenase that catalyzes the dehalogenation of dichloromethane (DCM) to formaldehyde and two equivalents of HC1, thereby allowing the bacteria to grow on D C M as sole carbon and energy source (Doronina et al. 2000) Similarly, OrfE3 of Sphingomonas sp. RW1 catalyzes the isomerization of maleylpyruvate, a catabolite of gentisate, to fumarylpyruvate (Vuilleumier et al. 2001). Nevertheless, the physiological role of most bacterial GSTs remains unknown. BphK occurs in some, but not all, bph pathways and, as noted above, does not appear to be essential for growth on biphenyl (Bartels et al. 1999). BphK of B. xenovorans LB400 catalyzes the GSH-dependent dehalogenation of 4-chlorobenzoate (4-CBA) (Gilmartin et al. 2003), a metabolite that is generated in the biotransformation of 4-C1 biphenyls. 4 - C B A can be transformed to protoanemonin, an antibiotic, by enzymes of the 3-oxoadipate pathway (Gilmartin et al. 2003). It has therefore been hypothesized that BphK was recruited by some bph pathways to assist in the degradation of PCBs. However, the specific activity of BphK for 4-C B A is quite low, approximately 30,000 times lower than for l-chloro-2,4-dinitrobenzene (CDNB) (Hofer et al. 1994), a reporter substrate that is often used to characterize GSTs. In light of this low activity, the physiological substrate of BphK, and the enzyme's role in the bph pathway, remain unclear. 1.7 Metabolic Blocks Although the bph pathway enzymes have evolved over millions of years to degrade biphenyl, and PCBs have only been present in the biosphere in significant amounts for less than a century, a surprising natural diversity exists in the PCB-transforming abilities of these enzymes. Thus, the range of PCBs that is transformed by the bph pathway varies in a strain-dependent manner: some bacterial strains are unable to transform PCBs containing more than 3 chlorines, whereas other strains, such as the Gram negative Burkholderia xenovorans LB400 previously Pseudomonas sp. LB400; (Bopp 1986; Kohler et al. 1988) and the Gram positive Rhodococcus RHA1 (Seto et al. 1995), transform up to hexachlorinated biphenyls. However, even these "potent" PCB-degraders can not effectively mineralize even lightly chlorinated congeners. A critical step in improving the PCB-transforming properties of bacterial strains is to determine the specificity of the Bph enzymes for P C B metabolites. Most studies concerning the microbial catabolism of PCBs have been carried out on B P D O , the first enzyme of the bph pathway, and considerable effort has been directed towards characterizing and engineering this enzyme (see section 1.8). BphB, the second enzyme of the pathway, was proposed to have a very broad substrate specificity, in part because it catalyzes the dehydrogenation of 3,4-dihydro-3,4-dihydroxy-2,2',5,5'-tetrachloro-biphenyl at a very low rate (Barriault et al. 1999). However, a subsequent study showed that it limits the degradation of some PCBs (Bruhlmann and Chen 1999). Studies using whole cells and purified enzyme indicate that D H B D , the third enzyme of the pathway, is critical in the degradation of some congeners. A n early study by Furukawa demonstrated that when resting cells of Alcaligenes sp. Y42 or Acinetobacter sp. P6 (now R. globerulus P6) were incubated in the presence of 2,6-dichlorobiphenyl or 2,2',5,5'-tetrachlorobiphenyl, DHBs accumulated in the culture medium, indicating that D H B D is unable to transform the expected metabolites: 2,6-dichloro D H B (2',6'-diCl D H B ) and 2,2',5,5'-tetrachloro 3,4-dihydroxybiphenyl (3,4-DHBs), respectively (Furukawa et al. 1979). Similarly, when a recombinant E. coli strain containing the bph pathway of LB400 was incubated with 2,6-diCl biphenyl and 2,5,3'-triCl biphenyl, D H B s accumulated suggesting that DHBDLB4OO is also unable to transform 2' ,6 '-diCl D H B (Figure 6A) and 3,4-DHBs (Figure 6B) (Seeger et al. 1995a). Biochemical studies using purified preparations of DHBDLB400 demonstrated that this enzyme cleaves chlorinated DHBs with lower specificity than unchlorinated D H B , is more 3-CI HOPDA species Figure 6. Metabolic blocks of the upper bph pathway. A ) 2 ' ,6 ' -diCl DHBs competitively inhibit D H B D . B) D H B D has no activity towards 3,4-DHB species. C) 3-CI H O P D A s competitively inhibit BphD. susceptible to oxidative inactivation during cleavage of chlorinated DHBs, and is competitively inhibited by 2' ,6 '-diCl D H B {Kic = 7 nM) (Dai et al. 2002). This molecule has a high affinity for the enzyme but the DHBD:2 ' , 6 ' -d iC l D H B complex reacts inefficiently with O2. A crystallographic structure of D H B D complexed with this metabolite suggests that the ortho chlorine substituents block the binding of the O2 to Fe 2 + , thus preventing the formation of the ternary complex (Dai et al. 2002). As mentioned earlier, D H B D is also potently inactivated by 3-CI catechol, originating from the degradation of certain congeners chlorinated at position 3 (Vaillancourt et al. 2002b). DHBDP6-I and DHBD P 6 - I I I , two evolutionarily divergent isozymes from the P C B -degrading R. globerulus P6 (Asturias and Timmis 1993; Asturias et al. 1994), have markedly different reactivities compared to DHBDLB40O (Vaillancourt et al. 2003). Indeed, DHBDp6-III, a one-domain enzyme, possessed greater specificity for some chlorinated D H B s than for D H B . However, D H B D P 6 - I and D H B D P 6 - I I I shared the relative inability of DHBDLB400 to cleave 2',6'-diCl D H B and 3,4-DHB. There have been no reports of successful attempts to overcome these metabolic blocks by engineering these enzymes. BphD is a bottleneck in the degradation of a different set of P C B congeners. Thus, strains Y42 and P6 transform PCBs possessing 2,4' chloro substituents to unidentified H O P D A s (Furukawa et al. 1979). Similar observations have been reported in several other strains, including B. xenovorans LB400 (Bedard and Haberl 1990; Seeger et al. 1995a). The corresponding chlorinated H O P D A molecules were later characterized as being 3-C1 H O P D A species (Seah et al. 2000). Seah et al. demonstrated that BphDLB400 hydrolyses H O P D A s chlorinated on the phenyl ring (positions 8-12) with very high specificity. However, this enzyme possesses very low activities against H O P D A s chlorinated on the dienoate moiety (positions 3-5; Figure 6C). Indeed, 3-C1 and 4-C1 H O P D A are hydrolysed 103 and 104 times slower than H O P D A (Seah et al. 2000; Seah et al. 2001). Similar activities have also been observed with a hydrolase from R. globerulus P6 (Seah et al. 2001). It has been hypothesized that this low activity is due to a reduced enzyme-catalysed enol-keto tautomerization required for the hydrolysis reaction (Henderson and Bugg 1997). 1.8 Unblocking the pathway The bph pathway appears to be inhibited by a relatively limited number of critical P C B metabolites. This suggests that the engineering of D H B D and BphD to transform key recalcitrant or inhibitory metabolites, or recruiting new enzymes active against these metabolites, might significantly enhance the PCB-degrading capabilities of microbial strains. Such strains might be particularly useful for two-stage treatment strategies in which an initial treatment with anaerobic strains reduces the complexity of P C B mixtures. Efforts to improve the PCB-degrading capabilities of natural isolates have included the engineering of bph enzymes to transform a broader range of congeners, and the introduction of downstream pathways to degrade the chlorobenzoates produced by the bph pathway (Gibson and Parales 2000). Most of the engineering has focused on the catalytic component of B P D O , the first enzyme of the pathway (Suenaga et al. 2001; Furukawa et al. 2004). The large subunit of B P D O harbors the principal determinants of the enzyme's substrate specificity. Accordingly, the corresponding genes from divergent bacterial strains have been subjected to D N A shuffling, generating variants that transform certain P C B congeners at higher rates (Kumamaru et al. 1998; Barriault et al. 2002). As might be expected, this focus on the first enzyme of the pathway is not a complete solution inasmuch as other bph enzymes are inhibited by certain P C B metabolites (vide supra). Strains that degrade other biaryl compounds potentially provide a useful source of P C B -transforming enzymes. For example, naphthalene and dibenzofuran are initially degraded by pathways that are very similar to the upper bph pathway. DoxG, an extradiol dioxygenase involved in naphthalene degradation by Pseudomonas sp. strain CI8 , catalyses the extradiol cleavage of 1,2-dihydroxynaphthalene (1,2-DHN) (Figure 7A; Denome et al. 1993). Interestingly, DoxG also catalyzes the cleavage of D H B , 3,4-DHB, and 2,2'5,5'-tetraCl 3,4-D H B , albeit with low specificity (Barriault et al. 1998). The extradiol cleavage of 3,4-DHB by D o x G yields a mixture of distally and proximally cleaved products, 2-hydroxy-4-phenyl-6-oxo-hexa-2,4-dienoic acid (4-phenyl H O D A ; Figure 7B) and 5-phenyl H O D A (Figure 7C), in a 2.8:1 ratio. Recent crystal structures of complexes of DoxG bound to 1,2-DHN, D H B and 3,4-DHB, respectively, revealed that the enzyme possesses a relatively large substrate pocket and binds these substrates in three different conformations (Neau 2004). Such a large substrate-binding pocket would be a good starting point for engineering experiments. Similarly, 2,2',3-trihydroxybiphenyl dioxygenase of Sphingomonas sp. RW1 (THBDRWI ) is involved in the Figure 7. Reactions catalyzed by DoxG. A ) Transformation of 1,2-dihydroxy-naphthalene (1,2-DHN) . B) Distal cleavage of 3,4-dihydroxybiphenyl occurs between C-4 and C-5 to form 2-hydroxy-4-phenyl-6-oxo-hexa-2,4-dienoic acid (4-phenyl H O D A ) . C) Proximal cleavage of 3,4-dihydroxybiphenyl occurs between C-2 and C-3 to form 2-hydroxy-5-phenyl-6-oxo-hexa-2,4-dienoic acid (5-phenyl H O D A ) . HOOC. OH 5-phenyl HODA degradation of dibenzofuran. The natural substrate of this enzyme is a structural analog of 2 ' -C l D H B (Happe et al. 1993). 1.9 Enzyme engineering by directed evolution Directed evolution has emerged as a powerful technique for the development of new biocatalysts. This technique harnesses the principles of natural evolution: generation of diversity by random mutagenesis and/or D N A recombination and positive selection of the small number of beneficially mutated genes (Brakmann et al. 2002). By applying this process iteratively, large changes in biocatalyst function can be achieved through the accumulation of small changes over many generations (Brakmann 2001). Contrary to structure-based, "rational" forms of protein engineering, directed evolution does not require detailed knowledge of the target enzyme's structure and function. The success of directed evolution experiments depends critically on both an effective exploration of sequence space and an efficient high-throughput screen to identify appropriately mutated enzymes. Figure 8 summarizes the directed evolution approach. 1.10 Exploring the sequence space: mutagenesis techniques and library creation A plethora of mutagenesis techniques has been developed in the past decade and new approaches continue to emerge. A l l mutagenesis techniques aim to create genetic diversity, ultimately to enable a simple and efficient exploration of sequence space for the desired enzymatic function (Brakmann 2001). Generally, these techniques involve cloning the target gene in a plasmid used for propagation and production of a library of mutants. Three fundamentally different types of mutagenesis techniques may be identified: random mutagenesis, saturation mutagenesis and D N A shuffling. The choice of mutagenesis technique is critical to the success of a directed evolution experiment. A Repeat cycle (optional) Protein expression Isolate corresponding gene variant Screen/selection for desired activity Protein variants Figure 8. General directed evolution strategy. Random mutagenesis usually targets whole genes and can be achieved in several ways. Perhaps the simplest involves propagating the plasmid D N A containing the target gene in a mutator strain, thereby generating a spectrum of single base substitutions, insertions and deletions (Greener et al. 1996). Random mutagenesis can also be accomplished by treating single stranded D N A with chemical mutagens (Kadonaga and Knowles 1985). Nevertheless, the most commonly used approach is error prone-PCR, where the fidelity of the D N A polymerase is modified by altering the concentrations of nucleotides, manganese and magnesium in the P C R reaction mixture (Cadwell and Joyce 1992). Mutation frequencies of up to 2% per base can be obtained. However, a typical mutation obtained by error-prone mutation involves only one nucleotide in a given codon. Consequently, the number of accessible amino acid substitutions is limited to an average of 5.7 per position (Miyazaki and Arnold 1999). Although the mutagenesis strategies described above do not allow for a complete exploration of the sequence space, they facilitate the identification of interesting regions or particular codons in the target gene. Saturation mutagenesis allows for a more complete exploration of the sequence space of a given D N A fragment. In this approach, D N A oligonucleotides are designed such that the codon specifying the target amino acid residue is degenerate, producing libraries that contain all possible 20 residues at that position (Miyazaki and Arnold 1999). Due to the enormous genetic diversity created by this technique, it can only be applied to a few residues at once. For example, saturation mutagenesis of 4 codons would produce a library o f - 1.7 x 10 7 clones. D N A recombination, or D N A shuffling, is a strategy that mimics genetic mating (Stemmer 1994). This approach is very effective in evolving new functions, presumably by concentrating beneficial mutations obtained separately or by recombining D N A fragments from homologous parental genes that have evolved independently (Crameri et al. 1998; Brakmann 2001). In an early experiment, Crameri et al. shuffled four homologous cephalosporinase genes together, obtaining a variant whose moxalactamase activity was 540-fold higher than that of any of the parental enzymes (Crameri et al. 1998). In the last few years, several D N A shuffling methods have been developed to overcome some of the limitations of the original method (Valetti and Gilardi 2004). In the Staggered Extension Process (StEP), a PCR-like reaction characterized by short elongation steps (typically 5-10 s) was developed to overcome the need for template fragmentation by DNAse l . Iterative Truncation for the Creation of HYbr id enzymes (ITCHY), was developed to obtain single crossovers between non-homologous genes, overcoming the limitation of using genes with over 85% sequence identity. A third approach, called R A C H I T T (Random CHImeragenesis on Transient Templates), is based on the hybridization of small single stranded D N A fragments on a complementary single stranded template D N A molecule (Coco et al. 2001). This technique increases the number of crossovers to an average of 14 per gene, producing highly recombined variants. D N A shuffling tends to be more effective when applied to highly homologous genes, and is especially powerful when used in combination with error-prone P C R and saturation mutagenesis (Brakmann 2001). 1.11 Biological selections and screens As critical as an efficient exploration of sequence space is to the success of a directed evolution experiment, the screen or selection used to identify the beneficially mutated clones is arguably the most critical aspect of a directed evolution experiment (Brakmann 2001). A screen usually requires the examination of all variants in the library whereas a selection strategy allows for the identification of the desired variants under conditions promoting their exclusive survival. For this reason, biological selections are generally perceived as the most powerful way to identify the desired enzyme variants. The major advantage of selections originates from the fact that libraries of ~10 1 0 clones can be examined, an upper limit imposed by the efficiency of cell transformation by D N A vectors (Taylor et al. 2001). They are usually based on complementation of auxotrophy or resistance to cytotoxic agents such as antibiotics. For example, Yano et al. increased the specificity of an aspartate aminotransferase for 2-oxovaline by 105-fold through several rounds of mutation and selection using an auxotroph for P-branched amino acids as a screening host (Yano et al. 1998). Nevertheless, such selection is limited by the ability to link the desired activity to the host cell viability. Fortunately, improved understanding of cellular physiology, especially sensory and signaling proteins, provides a growing number of ways to link activities to selectable phenotypes. Among sensory proteins, transcriptional regulators represent a particularly interesting class of systems for coupling the desired protein activity with cell viability as they control gene expression under precise physiological conditions. Placing an antibiotic resistance gene under the control of an engineered regulatory system that responds to a product of the desired enzymatic activity could potentially create a very powerful biological selection. Nevertheless, in vivo conditions are often difficult to predict and control, which can give rise to false positives. Selections can also be performed in vitro, as exemplified by phage display. Because host cell infection by filamentous bacteriophages is more efficient than transformation by plasmids, phage display typically allows for the examination of larger libraries, containing up to 10 1 2 clones (Smith and Petrenko 1997; Soumillion and Fastrez 2002). Briefly, the gene encoding the protein of interest is fused with a gene encoding a phage coat protein. The fusion gene is incorporated into the phage genome, and the encoded phage particle displays the protein of interest on its surface. Protein variants can then be extracted from a library on the basis of their ability to bind specific ligands. Phage particles that are attached to ligands are recovered and propagated in a host cell where the gene encoding the selected protein variant is identified and characterized. The power of phage display derives from the physical linkage of the gene and its corresponding protein by the phage particle (Soumillion and Fastrez 2002). To date, this approach has been most successful for selecting high-affinity ligands and receptors (Smith and Petrenko 1997). In contrast, few strategies have been developed for the selection of catalytically active enzymes (Pedersen et al. 1998; Jestin and Kaminski 2004). High-throughput screens are generally used to directly measure the activity of enzyme variants. This approach usually requires that the desired activity be coupled to a colorimetric assay. Screens are arguably more versatile than selections because the former can be performed in an artificial environment using substrates inaccessible to the growing cell. Such high-throughput screens are typically performed in microtiter plates using microplate readers (Brakmann 2001). However, even with the best high-throughput screens, significantly smaller libraries can be processed than with selections, throughput rates are typically ~10 5 clones per day (Taylorsal . 2001). 1.12 Aim of this study The overall objective of this doctoral research was to engineer and recruit enzymes to degrade critical P C B metabolites to improve the pollutant-degrading abilities of bacterial strains. This overall objective was pursued through three specific aims that focused on chlorinated D H B s and H O P D A s : (1) to characterize the ability of four evolutionarily divergent extradiol dioxygenases to transform specific chlorinated D H B s ; (2) to develop biological selections or high-throughput screens to facilitate the directed evolution of enzymes, particularly P C B -degrading ones; and (3) to investigate the role of BphK in P C B degradation. The four evolutionarily divergent extradiol dioxygenases that were studied were DHBDLB400, DHBDp6-I, DHBDp6-III and THBDRWI - Each enzyme was purified, and their respective abilities to cleave mono-, di- and trichlorinated D H B s were investigated. The susceptibility of each enzyme to oxidative inactivation during catalytic transformation of each D H B was also determined. Biological selections were engineered to couple the activities of D H B D and H O P D A hydrolase to cell viability. These selections were based on the detection of benzoate, an end product of the upper bph pathway. A n antibiotic resistance gene was placed under the control of a promoter activated by a transcription regulator recognizing benzoate as an effector molecule. For its part, the high-throughput screen is based on the colorimetric detection of H O P D A . The screen was used as a part of the directed evolution strategy to engineer an extradiol dioxygenase with a 3,4-D H B cleaving activity. This strategy combined error-prone PCR, saturation mutagenesis and D N A shuffling. The best enzyme variant was purified and characterized. Finally, the GST from the bph pathway, BphK, was purified and its ability to transform a range of P C B metabolites was investigated. Steady-state kinetics were conducted to determine the specificity of the enzyme for selected P C B metabolites and glutathione. The results are discussed and the physiological relevance of the observed activities is addressed. 2 MATERIALS AND METHODS 2.1 Chemicals A l l 2,3-dihydroxybiphenyls (DHBs) were synthesized according to standard procedures (Nerdinger et al. 1999) and were gifts from Victor Snieckus (Department of Chemistry, Queen's University, Kingston, Ontario). 1,2-DHN and C D N B were from Sigma-Aldrich (Oakville, Ontario, Canada). 2,6-Dichlorobiphenyl and 2,2',5,5'-tetrachlorobiphenyl were from Accustandard (New Haven, CT) . H O P D A , 3-CI H O P D A and 3,9,11-triCl H O P D A were produced enzymatically from D H B , 4-C1 D H B and 4,3',5'-triCl D H B , respectively, and purified by H P L C following procedures described previously (Seah et al. 2000). 3,4-DHB was from U L T R A Scientific (North Kingstown, RI). 2,2',5,5'-tetraCl-3,4-DHB was produced enzymatically from 2,2',5,5'-tetrachlorobiphenyl as described previously (Barriault et al. 1998). Ferene S was from I C N Biomedicals Inc. (Costa Mesa, C A ) . Restriction endonucleases and Antarctic Phosphatase were purchased from New England Biolabs (Beverly, M A ) . T4 D N A ligase and Taq D N A polymerase were from Amersham Biosciences (Baie d'Urfe, QC) and Promega (Madison WI), respectively. A l l other chemicals were of analytical grade and used without further purification. 2.2 Oligonucleotides and DNA sequencing The oligonucleotides used in this study were purchased from Operon Biotechnologies (Huntsville, A L ) or Integrated D N A Technologies (Coralville, IA) and are presented in Table 1. A l l D N A sequencing was performed on an A B I 373 Stretch (Applied Biosystems, Foster City, C A ) using Big-Dye v3.1 terminators at the Nucleic Ac id Protein Service Unit at the University of British Columbia. Table 1. Oligonucleotides used in this study Oligonucleotide Sequence8 Restriction site 5'RW1 AGTTATCCATATGTCAGTAAAACAA Ndel 3'RW1 CGCGGATCCTTCGTCACTACG BamHl 5K AAGCGCTAGCATGAAACTTTACTACAGCCC Nhel 3K GCGCAAGCTTCGGGAAGGCATCTTC BamVO. 5'doxG GAGATTCCATATGAGTAAGCAAGCTGCAGT Ndel 3'doxG CGGGATCCTTAGCTCAGTTTTACATCCAGGC BamHl T7For GACTCACTATAGGGAG oT7-7rev2 CTCATGTTTGACAGCTTATC KSDM AAAGATCAATGTTGACATAAGCGCTCCAACCGAGCAC 5Knde GCGATTCCATATGAAACTTTACTACAGCCC Ndei 242SM CGAAATTGACATTGCCNNKCAGCTTGGCATTCAGC 190-191SM GGCATGACTGCCGAANNKNNKTTCATGCATTGCAAC lOlsm GCGACAAAGTTGAGGCTNNKGAGCGTATGGTGTTGGG 154sm CTTGGGCCATTGCMNKGTTCGCCAAACCG The degenerated codons are shown in bold. N = A , T, C or G ; K = G or T. 2.3 Bacterial strains, plasmids and growth conditions Bacterial strains and plasmid vectors used in this study are described in Tables 2 and 3, respectively. Unless otherwise stated, Escherichia coli and Pseudomonas strains were grown at 37 °C and 30 °C, respectively, and shaken at 200 rpm in Luria Bertani (LB) broth with the appropriate antibiotics. E. coli strains DH5ct (Hanahan 1983), CC118Xpir (Herrero et al. 1990) and Nova Blue DE3 ( E M D Biosciences, Inc.) were used for D N A propagation and cloning. Protein was produced in Pseudomonas putida KT2442 (Herrero et al. 1990) and various E. coli strains. For P. putida KT2442, cells were transformed with derivatives of pVLT31 (de Lorenzo et al. 1993a) and cultured in L B supplemented with 20 (J.g/ml tetracycline, a potassium phosphate buffer described for terrific broth (Ausubel et al. 2000), and 10 ml of a mineral salts solution. The latter contained 14.1 m M M g C l 2 - 6 H 2 0 , 2.5 m M C a C 0 3 , 4.27 m M FeS0 4 -7H 2 0 , 0.625 m M Z n S 0 4 - 7 H 2 0 , 0.625 m M M n S 0 4 - H 2 0 , 0.125 m M C u S 0 4 - 5 H 2 0 , 0.125 m M C o C l 2 - 5 H 2 0 , 0.125 m M H 3 B 0 3 , 262.5 m M M g S 0 4 , 10 m M C a C l 2 , and 0.1 m M thiamine (Vaillancourt et al. 1998). Ten ml of an overnight culture were used to inoculate one liter of media in a 2-litre flask. Upon reaching an O.D. at 600 nm (OD6oo) of 0.5, the culture was supplemented with 0.5 m M of isopropyl-l-thio-p-D-galactopyranoside (IPTG) and was incubated for an additional 18 hours before harvesting. DOXGSMA2 was produced following the same protocol using E. coli Nova Blue DE3 transformed with pT7SMA2. BphK was produced using E. coli GJ1158 transformed with p T 7 B K l . Cells were grown in low salt L B (10 g tryptone extract, 5 g yeast extract per litre) supplemented with 100 ug/ml ampicillin. When the culture reached an OD600 of 0.5, N a C l was added to a final concentration of 0.3 M and the culture was incubated for an additional 12 hours before harvesting. Libraries of doxG variants were screened using E. coli Nova Blue DE3 and pT7-7 derivatives (Tabor and Richardson 1985). The libraries were grown at a density of 15-20 colonies/cm2 on solid medium containing 10% L B (1 g tryptone extract, 0.5 g yeast extract, 10 g N a C l and 15 g agar per liter), 0.5 m M IPTG and 100 ug/ml ampicillin. Table 2. Bacterial strains used in this study. Species and strains Relevant genotype or characteristics" Reference or origin Escherichia coli DH5a endAl, hsdR17, supE44, thi-1, X-recAl, gyr A96, relAl, AlacU169 (Hanahan 1983) (<j>801acZAM15) CCl 18 A(ara-leu), araD, AlacX74, galE, galK, phoA20, thi-1, rspE, rpoB, argE (Lee andManoil 1994) (am), recAl CC118Rif Rif derivative of CCl 18 This work CC118Xpir A(ara-leu), araD, AlacX74, galE, galK, phoA20, thi-1, rspE, rpoB, argE (Herrero et al. 1990) (am), recAl, Xpir lysogen; recipient of pCNBl derivatives (see plasmids below) S17.apir RecA, thi, pro, hsdRTVf, RP4::2-Tc::Mu::Km:: Tn7, Tpr Smr, >vpir (de Lorenzo et al. lysogen; mobilising strain for pCNB 1 derivatives 1993b) Nova Blue DE3 endAl hsdR17(TK\2- mK12+) supE44 thi-1 recAl gyrA96 relAl lac Novagen ¥"\proA+B+ laclgZAM15::Tnl0 (TcR)] GJ1158 proUpv.Tl KNAP::malQ-lacZ (Bhandari and Gowrishankar 1997) XylS Rif, Km', CCl 18Rif, mirii-Tn5: P„-c-xylS This work SHCl Rif, Km', Cm', CCl 18Rif, mini-Tn5: XylS-Pm-cat This work SHC2 Rif, Gm',Cm', CCl 18Rif, mini-Tn5: P„-c-xylS, mini-Tn5: Pm-cat This work SHC3 Rif, Cm', CCl 18Rif, mini-Tn5: Pm-xylS, mini-Tn5: Pm-cat This work SHC4 Rif, Tc', CCl 18Rif, mini-Tn5: Pm-xylS, mini-Tn5: Pm-tetC This work SHC 12 Rif, Gm', CC118Rif, mini-Tn5: P„c-xylS, mini-Tn5: Pm-luxCDABE This work Pseudomonas putida KT2442 HsdR, Rif prototrophic (Herrero et al. 1990) CL01 Rif, Km', KT2442mini-Tn5:/'«-x)'/5' This Work SHC 7 Rif, Tc', KT2442 mini-Tn5: Pm-tetC This Work SHC11 Rif, Gm', KT2442 mini-Tn5:/'m-/i«C£>.4.B£ This Work Sphingomonas sp. RW1 Dibenzofuran degrading strain. Lab stock 2 tnp* is the transposase gene of the IS50R of Tn5 devoid of its Notl sites in its DNA sequence (Herrero et al. 1990); Apr, ampicillin resistance; Cm', chloramphenicol resistance; Cms, chloramphenicol sensitive; Km', kanamycin resistance; Tcr , tetracyclin resistance; Tcs, tetracycline sensitive. Table 3. Plasmids used in this study Plasmids Relevant genotype, phenotype or characteristics2 Reference or origin pVLT31 Tcr, RSF1010-lacIq / Ptac hybrid broad host range expression vector, MCS of (de Lorenzo et al.) pUC18 pMPVDG Apr, doxG+ derivative of pVLT31 (Barriault et al. 1998) pAH92 Ap', pUC18 derivative containing bph pathway Lab stock pAHDl Apr, pVLT31, dbfB cloned as 1 Kb Xbal fragment from pT7dbfB This work pLEHP20 Apr, pEMBL18 derivatives containing his-tagged iso-1 from E halophila (Eltisera/. 1994) pT7HP20 Apr, pT7-7 containing the Xbal/Hindlll fragment from pLEHP20 This work pT7BIC20 Apr, pTHP20 contairming bphK as Nhel/Hindlll fragment This work pT7BK21 Apr, pTFTP20 contairming Ndel- mutant of bphK as Nhel/Hindlll fragment This work pT7-7 Apr, ori ColEl (Tabor and Richardson) pT7dbfB Apr, pT7-7, dbfB cloned as Ndel/C.BamHl fragment This work pT7doxG Apr, pT7-7, doxG cloned as Ndel/C.BamHl fragment This work pT7BKl Ap', pT7-7, bphK cloned as NdeVHindlU fragment This work pT7doxG242 Apr, encodes L242S variant of DoxG This work pT7doxG 190-1 Apr, encodes L190M-S191W variant of DoxG This work pT7SMA2 Apr, encodes SMA2 variant of DoxG This work pKK175-6 Apr, Tcs, source for promoterless tetracycline resistance gene (tetC) (Brosius) pKK232-8 Apr, Cms, source for promoterless chloramphenicol resistance gene (cat) (Brosius) pUC18Not Apr, pUC18 with MCS flanked by Notl sites (Herrero et al. 1990) pUC18Sfi Ap', pUC18 with MCS flanked by Sfil sites (Herrero et al. 1990) pUCGm Apr, Gmr, source for gentamycin resistance gene (aacCl) (Schweizer 1993) pUCSfiGm Apr, Gmr, gentamycin resistance gene (aacCl) flanked by Sfil sites This work pUCGmPm Apr, Gm', aacCl-Pm construct fanked by Sfil sites. This work pUCSfiPm Apr, Pm-containing -500 bp fragment flanked by Sfil sites This work pUCTet Apr, Tcr, contains the tetC gene flanked by Notl sites This work pPFCOl Apr, Cmr, pUC18Not, cat gene flanked by Notl sites This work pSB1075 Tcr, Contains luxCDABE as a C.BamWi fragment (Winson et al. 1998) pCNBl Apr, Km', ori RK6, mobRP4, tnp*, delivery plasmid for mini-Tn5, xylS/Pm (de Lorenzo et al. 1993 b) pCNBlCAT Apr, Km', Cmr, ori RK6, mobRP4, tnp*, delivery plasmid for mini-Tn5, xylS/Pm- This work cat pCNB5XylS Apr, Km', ori RK6, mobRP4, tnp*, delivery plasmid for mini-Tn5, lacf/P^-xylS pMP4 Apr, Gmr, ori RK6, mobRP4, tnp*, delivery plasmid for mini-Tn5, Pm-cat pUTPmCAT Apr, ori RK6, mobRP4, tnp*, delivery plasmid for mini-Tn5, Pm-cat pUTPT Apr, Tcr, ori RK6, mobRP4, tnp*, delivery plasmid for mini-Tn5, Pm-/erC pPFlux Apr, Gmr, ori RK6, mobRP4, tnp*, delivery plasmid for mini-Tn5, Pm-luxCDABE pBAD24 Ap', araC-?BAD pMOD Apr, colEI, EZ-Tn5™ Transposon construction vector pMAD Apr, 1.3 kb ClaVEcoKl fragment of pBAD24 ligated into the Clal/EcoPJ sites of pMOD pSS184 Ap', bphD+ derivative of pEMBL18, bphD cloned as Xbal/Hindlll fragment pMADbphD Ap', pMAD containing bphD as Xbal/Hindlll insert pMADBD Ap', pMADbphD containing dbfB as an Xbal insert pUCP24 Gm', E. coli-Pseudomonas shuttle vector pUCP24bphD Gm', pUCP24 containing bphD as XbaVHindlll insert pUCP24BD pUCP24bphD containing dbfB as an Xbal insert (de Lorenzo et al. 1993a) This work This work This work This work (Guzman et al. 1995) Epicentre (Madison, WI) This work (Seah etal. 1998) This work This work (West etal. 1994) This work This work " Apir is a A phage carrying the pir gene, the product of which (the n protein) is essential for replication of R6K plasmid and its derivatives, such as pCNBl (Herrero el al. 1990); Cm', chloramphenicol resistance; Km', kanamycin resistance; Rif, rifampicin resistance; Sm', streptomycin resistance; Tc', tetracyclin resistance; Tp', trimetoprim resistance. 2.3.1 Transformation and conjugation Transformation of E. coli and P. putida with plasmids was accomplished via electrotransformation (Nickoloff 1995). Conjugation was performed using the filter membrane mating technique (de Lorenzo and Timmis 1994), mini-Tn5 delivery vectors, E. coli S17.lX.pir as the donor strain and either E. coli CC118 (rif) or P. putida CL01 as the recipient strain. Briefly, the donor strain carrying the desired delivery plasmid was grown overnight at 37 °C in 2 ml of L B medium containing the antibiotics selecting for both the delivery plasmid and the mini-transposon. The recipient strains were grown overnight in 2 ml of L B medium without selection. The donor cells were harvested and washed twice with 2 ml of 1% N a C l to remove the excess antibiotics. Ten ul of the donor cells and 100 ul of the recipient cells were added to 5 ml of 1% NaCl , vortexed, then filtered through a 25 mm type H A 0.45 um Millipore membrane (Bedford M A ) . The filter was placed on L B solid medium and incubated at 30 °C for 12 hours. The filter was then immersed in 5 ml of 1% NaCl and vortexed to resuspend the mating culture. Exconjugants were selected by plating 10-500 ul aliquots of the mating resuspension on L B solid medium containing 1% citrate, 25 u.g/ml rifampicin and the antibiotic selecting for the mini-transposon. Successful transposition events were confirmed by testing for ampicillin sensitivity. 2.3.2 Cloning of the dbfB gene The gene encoding the THBDRWI, dbfB, was amplified by P C R from the chromosomal D N A of Sphingomonas sp. RW1 using two oligonucleotides: 5'RW1 and 3 'RW1. The P C R reaction was performed using the Pwo D N A polymerase (Roche Applied Sciences, Laval, P.Q., Canada) according to the manufacturer's instructions, with an annealing temperature of 45°C. The resulting amplicon was digested with Ndel and C.BamHl and cloned in pT7-7 to yield pT7dbfB. The cloned gene was sequenced to confirm its authenticity. The gene was then subcloned as anXbal fragment into pVLT31 , yielding p A H D l . 2.3.3 Cloning of the bphK gene The bphK gene was amplified by P C R from pAH92 using the primer pair 5K and 3K. The reaction was performed using the Expand High Fidelity™ D N A polymerase system (Roche Applied Sciences, Laval, P.Q., Canada) according to the manufacturer's instructions, with an annealing temperature of 45°C. The resulting amplicon was digested with Nhel and Hindlll and cloned into pT7HP20, yielding pT7BK20. A single Ndel restriction site present within the bphK gene was removed by site-directed mutagenesis using the Quick Change Multi™ mutagenesis kit (Stratagene, LaJolla, C A ) and the oligonucleotide K S D M , yielding pT7BK21. The bphK gene was amplified from pT7BK21 using the primers 5Knde and 3K. The resulting amplicon was digested with Ndel and Hindlll and cloned into pT7-7 (Tabor and Richardson 1985), yielding p T 7 B K l . The cloned bphK genes were sequenced to confirm their authenticity. 2.4 Construction of strains for Biological Selections and Screens (SHC systems) The following section details the construction of the host strains for biological selection and screen. In this thesis, the latter are referred to as selection host cells (SHC) systems. It also presents the construction of plasmids used for the characterization of the different selection systems. Figure 9 presents the pUT derivatives (de Lorenzo et al. 1990) that served as mini-Tn5 transposon delivery plasmids. 2.4.1 Cloning the reporter genes A l l reporter genes were cloned into pUC18Not to facilitate their subcloning into the pUT derivatives. 2.4.1.1 Cloning of the chloramphenicol acetyl-transferase (cat) gene The pKK232-8 plasmid (Brosius 1984) was digested with Earl, blunt-ended by Klenow fill-in, then digested with Xmal to release a ~1.6 kb D N A fragment containing the cat gene. Similarly, pUC18Not was digested with Hindlll, blunt-ended by Klenow fill-in, and digested with Xmal to produce a -2.6 kb D N A fragment. The latter was dephosphorylated. The two fragments were ligated to yield pPFCOl , a plasmid containing the promoterless cat gene flanked by Notl sites. 2.4.1.2 Cloning of the tetracycline resistance (tetC) gene The tetC gene was excised from p K K 175-6 (Brosius 1984) as a -1.5 kb Apol fragment and ligated into the EcoKl site of pUC18Not to yield pPFTOl , which contains the promoterless tetC gene flanked by Notl sites. 2.4.1.3 Cloning of the lux cassette The luxCDABE (lux) cassette was excised from pSB1075 (Middleton et al. 2002) as a - 6 kb C.BamRl fragment and ligated into the C.BamHl site of pUC18Not to yield pPFlux, which contains the promoterless lux cassette flanked by Notl sites. 2.4.2 Construction of S H C l and S H C 2 The cat gene from pPFCOl was excised as a -1.6 kb Notl fragment and ligated into the Notl site of p C N B l . The resulting plasmid, p C N B l C A T , contained the cat gene under the control of the Pm promoter (Figure 9). Proper orientation of the cat gene was confirmed by restriction analysis using Pstl, EcoRl and Ncol. E. coli S17.Rpir was transformed with p C N B l C A T and used for conjugation with E. coli C C l 18Rif to produce the biological selection host cell S H C l . SHC2 was constructed as follows. The gentamycin resistance gene (aacCl) was excised from p U C G m and cloned in pUC18Sfi as a -800 bp Sacl fragment to yield pUCSfiGm. pUCSfiGm contained aacCl in the reverse orientation relative to the Plac promoter of the plasmid. Subsequently, a -500 bp C.BamHl/Hindlll fragment containing the Pm promoter was excised from p C N B l and ligated to the -3.5 kb CBamUVHindm fragment of pUCSf iGm, to the -6.6 kb Sfil fragment of p C N B l C A T . Proper orientation of the ligated D N A fragments was Notl Notl —1> kmr H ^ - facf *WS pCNBlCAT Notl Notl —I aacCf cat Notl Notl me: pMP4 pUTPT Notl Notl —I aacCf —D^n>— 4 luxCDABE *i_L pUTlux Figure 9. Organization of the mini-transposons used to construct the SHC strains. The upper part shows the mini-Tn5 transposon constructs. The lower part shows the universal pUT-based mini-Tn5 delivery plasmid. Refer to table 3 for additional details. confirmed by restriction analysis using Xbal. The resulting plasmid, pMP4 (Figure 9), was used to produce SHC2 by conjugation between E. coli S17.1A,pir (pMP4) and E. coli X y l S . 2.4.3 Construction of SHC4, SHC7, S H C l 1 and SHC 12 The ~1.5 kb Notl fragment of pUCTet was excised and ligated to the ~6 kb Notl fragment of pUTPmCAT, yielding pUTPT (Figure 9). E. coli S17 .Upir (pUTPT) was used for conjugation with E. coli Xy lS and P. putida CL01 to produce SHC4 and SHC7, respectively. Finally, the -6 kb Notl fragment from pPFlux was ligated to the -6.7 kb Notl fragment of pMP4, yielding pUTlux (Figure 9). Conjugation of E. coli S17.lA.pir (pUTlux) with E. coli X y l S and P. putida CL01 produced SHC 12 and S H C l 1, respectively. 2.4.4 Cloning dbfB and bphD in pMAD and pUCP24. The genes dbfB and bphD (encoding the H O P D A hydrolase from B. xenovorans LB400) were cloned into the expression vectors p M A D and pUCP24. p M A D was constructed by ligating the -1.3 kb Clal/EcoW fragment of pBAD24 (Guzman et al. 1995) to the -2.5 kb Clal/EcoRl fragment of p M O D (Epicentre, Madison, WI) and contains a multiple cloning site located downstream of the arabinose-inducible PBAD promoter. The pUCP24 plasmid is an E. coli/Pseudomonas shuttle vector that contains a multiple cloning site located downstream of the IPTG-inducible P ] a c promoter (West et al. 1994). The -1.1 kb Xbal/Hindlll fragment from pSS184 (Seah et al. 1998) was excised and ligated to the -3.8 kb Xbal/Hindlll fragment of p M A D to yield pMADbphD. The same -1.1 kb fragment was also ligated to the -4 kb Xbal/Hindlll fragment of pUCP24 to produce pUCP24bphD. The -1 kb Xbal fragment of pT7dbfB was excised and also ligated to two vector fragments: the 4.9 kb Xbal fragment of pMADbphD and the 5.1 kb Xbal fragment of pUCP24bphD. This produced p M A D B D and pUCP24BD, respectively. The proper orientation of each construct was confirmed by restriction analysis using Pvull and Notl. 2.5 Expression tests of the reporter genes in the SHC systems The expression of all reporter genes was tested using both the donor strain E. coli S17.lX.pir containing the mini-Tn5 delivery constructs and the corresponding SHC systems. For the strain containing the cat or tetC genes under the control of the Pm promoter, inocula of 200-400 cells were plated on L B solid medium containing the selection antibiotics and 0-5 m M benzoate. The same concentrations of benzoate were used for the strains containing the lux cassette. Luminescence (relative light units) was detected on solid media using a LB980 photon camera (Berthold U S A , Oakridge, TN) and in liquid culture using microtiter plates and a 1420 Victor multilabel reader (Perkin-Elmer Life Sciences, Woodbridge, Ontario, Canada). For luminescence assays carried out in liquid cultures, 200 ul aliquots of mid-log phase cells grown in L B were added to a 96-well microtiter plate and incubated in the presence of 0 to 100 u M benzoate. The development of luminescence was followed in the multilabel reader for 4 hours, collecting measurements every 10 minutes. Cultures incubated in the absence of benzoate served as reference. The response of the SHC systems to the in vivo production of benzoate was investigated essentially as described above by incubating the SHC strains, transformed with pUCP24BD or p M A D B D , with D H B concentrations ranging from 0 to 500 u M . 2.6 Directed evolution ofdoxG: mutagenesis procedures The doxG gene was mutated for directed evolution using three approaches that were applied successively: 1) error-prone PCR; 2) saturation mutagenesis at selected positions; and 3) D N A shuffling. Libraries generated using each approach were transformed into E. coli Nova 33 Blue DE3 and screened for increased 3,4-DHB cleavage activity following the protocol described below. Libraries were constructed using doxG cloned into pT7-7. Briefly, doxG was amplified from p M P V D G (Barriault et al. 1998) using the primer pair 5'doxG and 3'doxG, Pwo D N A polymerase (Roche Applied Sciences, Laval, P.Q., Canada) according to the manufacturer's instructions, and an annealing temperature of 45 °C. The resulting amplicon was digested with Ndel and C.BamHl and cloned in pT7-7, yielding pT7doxG. The gene was sequenced to confirm its authenticity. 2.6.1 Error-prone PCR Error-prone P C R was carried out using Taq polymerase with pT7doxG as template and T7For and oT7-7rev2 as primers following published protocols (Cadwell and Joyce 1992). Accordingly, a 30-cycle P C R reaction was performed in the presence of 0.5 m M manganese chloride, 200 u M of dATP and dGTP, and 1 m M of dCTP and dGTP. The resulting amplicon was digested with Ndel and BamHl and cloned in pT7-7 to yield a library of doxG variants (see section 2.7 for cloning conditions). 2.6.2 Saturation mutagenesis Saturation mutagenesis was performed using the QuikChange® Multi Site-Directed Mutagenesis Ki t (Stratagene, La Jolla, C A ) and oligonucleotides containing 32-fold degenerated codons (NNG/T) at the target positions (Table 1). 2.6.3 DNA shuffling D N A shuffling was performed using the Expand High Fideli ty '" 5 polymerase mixture (Roche Applied Sciences, Laval, P.Q., Canada) according to published protocols (Stemmer 1994) using the conditions described below. The target genes were amplified using the Expand High Fidelity 7"" polymerase mixture according to the manufacturer's instructions using a 25 cycle P C R reaction and an annealing temperature of 45 °C to yield ~ 3 u.g of P C R products. The resulting amplicons were purified from agarose gel using the G F X - P C R gel purification kit (Amersham Biosciences, Baie d'Urfe, P.Q., Canada) and desalted using a Centri-Sep column (Princeton Separations, Adelphia, N.J.). The amplicons were treated with DNAse I to yield D N A fragments of 50-300 bp in size. The latter were separated by electrophoresis using a 2% agarose gel and the 50-200 bp fragments were purified using the following protocol. The D N A fragments of interest were excised from the gel and passed through a 22 gauge needle using a 1 ml syringe. Two volumes of a solution of 0.5 M ammonium acetate, 10 m M magnesium acetate hexahydrate, 1 m M E D T A , p H 8.0 were added to the shredded gel. The mixture was submitted to three successive freeze/thaw cycles. Each cycle consisted of 10 min incubations at 50 °C and -20 °C, respectively. The mixture was centrifuged for 10 minutes at 16 000 x g in a 0.22 urn Spin-X filter (Corning Inc. Life Sciences, Acton, M A ) . The D N A was ethanol-precipitated using standard protocols (Ausubel et al. 2000), resuspended in water and desalted using a Centri-Sep column. The final solution contained -10 ng/ul of D N A fragments. To 35 ul of this solution, 10 ul of the 5X reaction buffer provided with the Expand High Fidel i ty ' 1 " polymerase, 4 ul of 2.5 m M dNTPs mix, and 1 ul of the Expand High Fideli ty '" 5 polymerase (3.5 Unit/ul) were added. The reaction mixture was submitted to 35 cycles that consisted of 30 s at 95 °C, 5 min at 55 °C and 2 min at 72 °C in a Robocycler (Stratagene, L a Jolla, C A ) . After reassembly, the shuffled variants were amplified using the 5'doxG and 3'doxG primers as described above. The obtained amplicon was digested using Ndel and C.BamHl and cloned into pT7-7 (see section 2.7 for cloning conditions). 2.6.4 DoxG L242S and L190M/S191W variants The L242S variant was constructed by ligating the -600 bp Ndel/Bcll fragment of pT7doxG to the -2.8 kb Ndel/Bcll fragment of the vector encoding the S M A 2 variant (vide infra). Similarly, the L190M/S191W double variant was obtained using the -2.8 kb Ndel/Bcll fragment of pT7doxG and the -600 bp Ndel/Bcll fragment of the vector encoding S M A 2 . 2.7 Cloning conditions and library construction The libraries of variants obtained by error-prone P C R and D N A shuffling were produced by ligating the Ndel and C.itamHI-digested variant amplicons to the pT7-7 expression vector digested with the same restriction enzymes. To maximize the efficacy of the ligation and minimize the background transformants, all D N A fragments were purified from agarose gel and their restriction digestions were performed for a minimum of 8 hours using a concentration of endonuclease 4 times greater than recommended by the manufacturer. The vector D N A was dephosphorylated using Antarctica Phosphatase according to the manufacturer's instructions. A l l ligations were performed in a final volume of 15 (xl using a total D N A concentration of -15 ng/ul, an insert:vector molecular ratio of 3-5:1 and a j / E i ratio of 1.3-1.8 (Dugaiczyk et al. 1975). A l l ligation mixtures were incubated at 15 °C for 12 hours. 2.7.1 Determination of the required library size The size of the library needed was determined using the following equation: ln(l - P) n —-l n ( l - / ) Where P is the probability of coverage and / is the frequency of variation (Sambrook et al. 2000). 2.8 Colorimetric screen Libraries of DoxG variants (pT7-7 derivatives) were transformed into E. coli Nova Blue DE3, plated on 15-cm petri dishes and grown for 16 hours. Colonies were then sprayed with a 5 m M solution of 3,4-DHB. Those containing an extradiol 3,4-DHB cleaving activity developed a yellow coloration. To facilitate the detection and quantification of the resulting degradation products (X m a x = 400 nm), images of the plates were captured through a 400 nm optical filter at different time points using a Canon Powershot A80 digital camera (Canon Canada, Mississauga, Ontario, Canada) (Figure 10A). The blue channel of the picture was extracted using Photo Paint Shop Pro 9 (Corel Corporation, Ottawa, Ontario, Canada). In the resulting images, the colonies producing yellow products appeared as dark spots against a light background (Figure 10B). These spots were quantified using ImageQuant 5.2 (Amersham Biosciences). The colonies showing the highest pixel density were picked and grown in 96 deep-well plates in 1 ml L B medium containing 0.5 m M IPTG and 100 mg/1 ampicillin. 2.9 Activity assays in 96 well microplates The cells were harvested, frozen at -80°C for at least 15 minutes and lysed using BugBuster™ (Novagen, San Diego, C A ) . Activities were measured in the cleared cell lysates using 100 u M 3,4-DHB and potassium phosphate buffer pH 7.0 (1=0.1 M ) . Reactions were performed at 25 °C and the formation of ring-cleaved product was monitored at 405 nm using a Victor 1420 Multilabel microplate reader equipped with a two channel injector module (Perkin-Elmer Life Sciences, Woodbridge, Ontario, Canada). Upon injection of the substrate, the enzymatic reactions were followed in a single well at a rate of 5 reads per second for 20 seconds. The cleavage of D H B was monitored under similar conditions at 450 nm. Activities were calculated by measuring the initial slope of each assay. Raw extracts containing the wild-type enzyme were added to four wells in the microplates, which served as references to calculate the relative activity of each variant. Activities were normalized according to the expression level of each D o x G variant. F igure 10. Imaging strategy used to facilitate colony screening. A ) Colonies grown to a density of 15-20 cfu/cm 2 were sprayed with a 5 m M solution of 3 ,4-DHB. The images were captured through a blue filter (max. transmission at 450 nm). B) Filtered image was processed in Corel Photo Paint Shop Pro8 to extract the blue colour channel. The active clones appear as dark spots on a light background. The pixel density (pixel/cm 2) corresponding to each active clone was determined using the Pharmacia ImageQuant software. Expression levels were assessed as follows. Aliquots of raw extracts corresponding to the variant of interest were loaded on a 12% S D S - P A G E . After electrophoresis, the gels were stained with SyproRuby and scanned using a Typhoon 9400 variable mode imager (excitation 488 nm, emission 610 nm; Amersham Biosciences, Baie d'Urfe, P.Q., Canada). The images were analyzed using ImageQuant 5.2 (Amersham Biosciences). The activity of each variant was corrected according to the intensity of the band corresponding to D o x G (-32.5 KDa) . 2.10 Protein purification Unless otherwise specified, all manipulations involving the dioxygenases were performed under an inert atmosphere in an Mbraun Labmaster 100 glovebox (Newburyport, M A ) maintained at 2 ppm Oj or less. Buffers were prepared and made anaerobic as described previously (Vaillancourt et al. 1998; Vaillancourt et al. 2003). A l l chromatography steps were performed on an A K T A Explorer (Amersham Biosciences, Baie d'Urfe, P.Q., Canada) configured to maintain an anaerobic atmosphere during purification, as previously described (Vaillancourt et al. 1998). DHBD L B 4oo, D H B D P 6 - I , and D H B D P 6 - I I I and DoxG enzymes were purified to apparent homogeneity following published protocols (Vaillancourt et al. 1998; Vaillancourt et al. 2003). A l l manipulations involving BphK were performed in air-saturated buffer. Protein purity was estimated by S D S - P A G E , which was performed in a Bio-Rad M i n i P R O T E A N II apparatus and stained with Coomassie Blue according to established procedures (Ausubel et al. 2000) 2.10.1 Purification of T H B D R W I Cells (P. putida KT2442 (pAHDl) ) from 4 litres of culture were harvested by centrifugation and frozen at -80 °C. The cell pellet (approximately 28 g) was resuspended in 36 ml of 50 m M phosphate, pH 7.5, containing 1 m M M g C l 2 , 1 m M CaCb, and 0.1 mg/ml DNase I. The cells were disrupted by three successive passages through a French Press (Spectronic Instruments Inc., Rochester, N Y ) operated at a pressure of 20,000 p.s.i. The cell debris was removed by ultracentrifugation in gas-tight tubes at 37,000 rpm for 90 min in a T1250 rotor (DuPont Instruments, Wilmington, DE) . The clear supernatant was carefully decanted and filtered using a 0.45 u M filter (Sartorius A G , Gottingen, Germany). This fluid was referred to as the raw extract. The raw extract was brought to 20% saturation ammonium sulfate and divided into four equal portions (~16 ml each). Each portion was loaded onto a Source™15 Phenyl (Amersham Biosciences) hydrophobic interaction column ( 2 x 8 cm) previously equilibrated with 50 m M phosphate pH 7.5, 2 m M dithiotreitol (DTT) (buffer A ) containing 0.85 M ammonium sulfate. Following the injection of T H B D R W I , the column was rinsed with 2 column volumes of buffer A containing 0.85 M ammonium sulfate and 3 column volumes of buffer A containing 0.68 M ammonium sulfate. THBDRWI activity was eluted using a linear gradient of 0.68 M to 0 M ammonium sulfate in buffer A (8 column volumes). Fractions of 8 ml were collected. Those containing enzyme activity were pooled and equilibrated with 10 m M Tris-Cl p H 7.5, 10% isopropanol, 2 m M DT T (buffer C) by three rounds of dilution/concentration using a stirred cell concentrator equipped with a Y M 1 0 membrane (Amicon, Oakville, Ontario, Canada). The resulting preparation (~10 ml) was divided into three parts, each of which was loaded onto a HR16/10 MonoQ anion exchange column (Amersham Pharmacia Biotech) equilibrated with buffer C. THBDRWI activity was eluted at a flow rate of 6 ml/min with a 15 column volume linear gradient of 0 to 250 m M N a C l in buffer C. Activity-containing fractions from the three runs were pooled, concentrated to 3 ml by ultrafiltration, and loaded onto a HiLoad 26/60 Superdex 200 column (Amersham Biosciences) equilibrated with buffer C containing 50 m M NaCl and 0.25 m M ferrous ammonium sulfate. The protein was eluted at a flow rate of 2.5 ml/min. Activity-containing fractions (5 ml) were pooled, concentrated to 22 mg/ml protein, and frozen as beads in liquid N2. Purified THBDRWI was stored at -80 °C for up to six months without any significant loss of activity. 2.10.2 Purification of DOXGSMA2 DOXGSMA2 was purifed from E. coli Nova Blue DE3 freshly transformed with pT7SMA2. The raw extracts were prepared as presented in section 2.10.1 except that the cell pellets were resuspended in 10 m M Tris, 10% glycerol, p H 7.5 (buffer A) . The raw extract was divided into two equal portions (~15 ml each). Each portion was loaded onto an HR16/10 MonoQ column equilibrated with 10 m M Tris, 10% glycerol, pH 7.5. The enzyme was eluted at a flow of 6 ml/min using a 320 ml linear gradient from 150 to 350 m M N a C l in buffer A . Fractions of 8 ml were collected. Activity-containing fractions were pooled, concentrated to 3 ml by ultrafiltration and loaded onto a HiLoad 26/60 Superdex 200 column equilibrated with buffer A containing 33 m M ammonium sulfate and 0.25 m M ferrous ammonium sulfate. The protein was eluted at a flow rate of 2.5 ml/min. Activity-containing fractions (5 ml) were pooled, concentrated to > 25 mg/ml protein, and frozen as beads in liquid N2. Purified DOXGSMA2 was stored at -80 °C for up to six months without any significant loss of activity. 2.10.3 Purification of BphK BphK was purified by affinity chromatography using the Glutathione Sepharose 4B resin (Amersham Biosciences, Baie d'Urfe, P.Q., Canada) according to the manufacturer's instructions. Briefly, cells from 1 liter of culture were harvested by centrifugation. The cell pellet (approximately 6 g) was resuspended in 10 ml PBS buffer (140 m M NaCl , 2.7 m M KC1, 10 m M N a 2 H P 0 4 , 1.8 m M KH2PO4, p H 7.3). The cells were disrupted by three successive passages through a continuous flow French Press (Spectronic Instruments Inc., Rochester, N Y ) operated at a pressure of 10,000 psi. The cell debris was removed by ultracentrifugation at 37,000rpm for 30 min in a T1250 rotor (DuPont Instruments, Wilmington, DE) . The clear supernatant was carefully decanted and filtered using a 0.45 um filter (Sartorius A G , Gottingen, Germany). This fluid was referred to as the raw extract. The raw extract was applied to the Glutathione Sepharose column (20 x 32 mm), washed three times with 100 ml of PBS buffer then eluted with 20 ml of 50 m M Tris-Cl p H 8.0, 10 m M G S H . The protein was concentrated to >50 mg/ml protein using a stirred-cell concentrator equipped with an Y M 1 0 membrane (Amicon, Oakville, Ontario, Canada) and frozen as beads in liquid N 2 . Purified BphK was stored at -80 °C for up to six months without any significant loss of activity. 2.11 Handling and characterization of dioxygenase samples Aliquots of dioxygenase were thawed immediately prior to use and were exchanged into 20 m M HEPPS, 80 m M N a C l (I = 0.1), 2 m M dithiotreitol, pH 8.0 by gel filtration chromatography (Vaillancourt et al. 1998). Samples of dioxygenase were further diluted using the same buffer supplemented with 0.1 mg/ml B S A . Protein concentrations were determined by the Bradford method (Bradford 1976). Iron concentrations were determined colorimetrically using Ferene S (Haigler and Gibson 1990). 2.11.1 Extradiol dioxygenases: steady-state kinetic experiments Experiments were performed using potassium phosphate buffer pH 7.0, I = 0.1 M , 25.0 ± 0.1 °C (290 u M dissolved 0 2 ) unless otherwise stated. The standard activity assay was performed spectrophotometrically using 100 u M D H B . Concentrations of active dioxygenase in the assay were defined by the iron content of the injected purified enzyme solution and were used in calculating kinetic parameters, including specificity, catalytic and inactivation constants. Steady-state rate equations (either the Michaelis-Menten equation or an equation describing a mechanism in which substrate inhibition occurs (Vaillancourt et al. 1998) were fit to data using the least squares and dynamic weighting options of L E O N O R A (Cornish-Bowden 1995). For specificity experiments in which the concentrations of DHBs and O2 were varied, the initial velocities, obtained by following the consumption of oxygene using a Clark-type electrode, were fit to the steady-state rate equation describing a compulsory order, ternary complex mechanism. One unit of enzymatic activity was defined as the quantity of enzyme required to consume 1 ^mol of 0 2 per minute 2.11.2 Spectrophotometric assay of ring-cleavage reaction The ring-cleavage reaction was also monitored by following the formation of H O P D A using a Cary 2 spectrophotometer (Varian, Palo Alto, C A ) equipped with a thermojacketed cuvette holder. The amount of enzyme used in each assay was adjusted so that the progress curve was linear for at least 1 min. Initial velocities were determined from a least-squares analysis of the linear portion of the progress curves using the kinetics module of the Cary W i n U V software. 2.11.3 Determination of partition ratio and inactivation constants Partition ratios were determined using the spectrophotometric assay described above and saturating substrate concentrations (i.e., the concentration of D H B exceeded Kapp by at least 15 fold). The amount of enzyme added to the reaction cuvette was such that the enzyme was completely inactivated before 15% of either the catecholic substrate or O2 was consumed in the reaction mixture. The partition ratio was calculated by dividing the amount of product formed by the amount of active D H B D added to the assay. The progress curves from these same reactions were also used to evaluate the apparent rate constant of inactivation during catalytic turnover in air-saturated buffer, jfp (Vaillancourt et al. 2002b). Under the assay conditions, the concentration of free enzyme, [E], is negligible and the partition ratio is equal to the ratio of the catalytic constant k°pf to the inactivation constant HPP . No. of substrate molecules consumed k, partition ration = = —— (1) No. of enzyme molecules inactivated £ ji For 2',6'-diCl D H B , j°pp was evaluated from the rate of inactivation js using equation 2. The latter was determined from progress curves obtained spectrophotometrically at 391 nm from reactions performed at saturating substrate concentrations [S] (100 to 200 uM) by using equation 3. . J°PP[S] L KT+[S] ( 2 ) P,=P„(\-e-J-') + P, (3) In equation 2, K^ppis the apparent Km of 2',6'-diCl D H B in air-saturated buffer. In equation 3, P , and PQC, are the concentrations of product at the start and end of the assay, respectively, and Pt is the concentration of product formed at time t. 2.11.4 H P L C analysis of cleavage products The reaction products of the DoxG-catalyzed cleavage of 3,4-DHB were separated by reverse-phase chromatography using a Waters 2695 separation module equipped with a Waters 2996 photodiode array detector and a Cis Symmetry 3.5 um column (4.6 x 75 mm) (Waters, Mississauga, Ontario, Canada). After the enzymatic reaction, acetonitrile and phophosphoric acid were added to final concentration of 20% and 0.5%, respectively. The resulting solutions were centrifugated at 16 000 x g for 2 minutes and aliquots of 50 ul of the supernant were injected on the H P L C . Products were eluted using a 20% to 90% acetonitrile gradient in an aqueous solution of 0.5% phosphoric acid at a flowrate of lml/min. Elution of the products was monitored at 254 ran. 2.12 Handling and characterization of BphK samples Aliquots of BphK were thawed immediately prior to use and were diluted in potassium phosphate buffer, pH 6.5 (I = 0.05 M ) , supplemented with 0.1 mg/ml B S A . Protein concentrations were determined by the Bradford method (Bradford 1976) and by measuring the absorbance of the protein solution at 280 nm using an extinction coefficient obtained from amino acid analysis (s = 33,300 IVf'cm"1). 2.12.1 Ability of BphK to transform PCB metabolites The activity of BphK towards 4-chlorobenzoic acid (4-CBA), 4-C1 D H B , 5-C1 D H B , 6-C l D H B , H O P D A and 3-CI H O P D A and 3,9,11-triCl H O P D A was assessed in potassium phosphate buffer, pH 6.5 (I = 0.05 M ) at 25.0 ± 0 . 1 °C supplemented with 1 m M G S H . A l l assays were performed using 100 to 2500 u M of P C B metabolites and 20 to 2000 u M enzyme. Aliquots of the reaction were withdrawn periodically and analyzed by H P L C using the Waters 2695 separation module and a Cis Symmetry 3.5 urn column (4.6 x 75 mm) (Waters, Mississauga, Ontario, Canada). The reaction products were eluted using a 60:40 mixture of methanol:water at a flow rate of 1 ml/min. The inorganic chloride ion concentration was measured using a published procedure (Bergmann and Sanik 1957). The concentration of reduced G S H was determined using 5,5'-dinitrothiobenzene (Ellman 1959). 2.12.2 Evaluation of BphK steady-state kinetic parameters The steady-state activity of BphK was measured spectrophotometrically using a Varian Cary 2 equipped with a thermojacketed cuvette holder. The standard assay conditions used potassium phosphate buffer, p H 6.5 (I = 0.05 M ) at 25.0 ± 0 . 1 °C and 1 m M of G S H . Assays performed using 1 m M C D N B were monitored at 340 nm as previously described (Habig and Jakoby 1981). Assays performed using H O P D A or 3-C1 H O P D A were monitored at 433 or 438 nm, respectively. One unit of enzymatic activity was defined as the quantity of enzyme required to consume 1 umol of substrate per minute. The amount of enzyme used in each assay was adjusted so that the progress curve was linear for at least 1 min. Initial velocities were determined from a least-squares analysis of the linear portion of the progress curves using the kinetics module of the Cary software. The steady-state kinetic parameters for 3-C1 H O P D A and 3,9,11-triCl H O P D A were determined using substrate concentrations ranging from 4 to 100 u M and an enzyme concentration of -0.04 u M . The apparent Km for G S H was determined using a concentration of 50 u M 3-C1 H O P D A and 75 u M 3,9,11-triCl H O P D A and 0.02 to 10 m M G S H . The Michaelis-Menten equation was fit to data using the least squares and dynamic weighting options of L E O N O R A (Cornish-Bowden 1995). For specificity experiments in which the concentrations of both chlorinated H O P D A s and G S H were varied, the initial velocities were fit to the steady-state rate equation describing a ternary complex mechanism without substrate inhibition. 2.12.3 Characterization of the BphK-catalyzed transformation product of 3-C1 HOPDA A reaction was performed in 200 ml of 50 m M potassium phosphate buffer, p H 6.5 at 25 °C containing 20 umol of 3-C1 H O P D A , 200 umol of G S H and 100 nmol of BphK. The reaction was allowed to proceed to >80% completion as determined spectrophotometrically. BphK was removed by ultrafiltration using a stirred cell equipped with a Y M 1 0 membrane (Amicon). The enzyme-free ultrafiltrate, which contained >80 u M o f the reaction product, was acidified to pH 3 by the addition of 200 ml of an aqueous solution of 0.5% orthophosphoric acid. The resulting solution was extracted twice with ethyl acetate. Upon ethyl acetate evaporation using a rotary evaporator, the product was purified by H P L C using a Waters 2695 separation module and a Phenomenex Prodigy 10-um ODS-Prep column (21.2 x 250 mm) (Phenomenex, Torrance, C A ) . The column was eluted with a 50:50 mixture of methanol:water at a flow rate of 7 ml/min. The retention time of the product was -40 minutes. The fractions containing the product were pooled, added to 4 volumes of aqueous 0.5% orthophosphoric acid then extracted as described above. The p K a of the compound was determined according to published protocols (Seah et al. 2000). The product was further characterized by U V - V i s Spectrophotometry and analytical H P L C as described in section 2.12.1. 2.13 Protein structure modeling of T H B D R W I The structure of THDBRWI was modeled using M O D E L L E R 6v2 (Sali and Blundell 1993) and the crystallographic coordinates of DHBDLB400 bound to 2 ' ,6 ' -diCl D H B (PDB id: 1 L K D ; Dai et al. 2002 ). The amino acid sequences of THBDRWI and DHBDLB400 were aligned using T-C O F F E E (Notredame et al. 2000). The fitting and minimization routines of M O D E L L E R were performed using the default parameters. 3. RESULTS 3.1 Transformation of PCB metabolites by evolutionarily diverse extradiol dioxygenases The reactivities of three two-domain type I extradiol dioxygenases (THBDRWI, DHBDLB400 5 DHBDP6-I ) and a one-domain type I extradiol dioxygenase (DHBDp6-III), towards mono-, di- and trichlorinated 2,3-dihydroxybiphenyls (DHBs) were investigated. Steady-state kinetic and inactivation studies were conducted to determine the apparent specificities of the enzymes for each D H B and their susceptibility to mechanism-based inactivation. 3.1.1 Purification of T H B D R W I and other DHBDs THBDRWI was purified anaerobically using a two-column procedure. Relevant details are summarized in Table 4. Over 100 mg of THBDRWI was purified from 4 L of cell culture to >98% apparent homogeneity as judged by S D S - P A G E (data not shown). The purified enzyme contained greater than 95% of its complement of iron and had a specific activity for D H B of 108 U/mg. Preparations of the three D H B D s were of previously reported quality (Vaillancourt et al. 1998; Vaillancourt etal. 2003). Table 4. Purification details of THBD] 'RWl Enzyme and purification step Total protein Total activity Specific Activity Yield (mg) (U) (U/mg) (%) Raw extract 2700 70300 27 100 Source 15 Phenyl 250 20500 82 29 Superdex 200 110 12000 110 17 Activity units are defined in section 2.11.1. 3.1.2 Physical properties of HOPDAs The extinction coefficients of chlorinated H O P D A s were determined to enable the spectrophometric evaluation of the reactivity of D H B D s with chlorinated DHBs. Solutions of the freshly prepared HOPDAs , produced from the DHBD-catalyzed cleavage of the corresponding D H B s (Figure 11) had the characteristic yellow color of the enolate anion. Accordingly, the absorption spectra of these solutions had wavelengths of maximal absorbance between 392 and 438 nm. The extinction coefficients at the corresponding wavelength for each H O P D A in 100 m M phosphate, pH 7.0 and 50 m M phosphate, pH 6.5, both at 25 °C, are given in Table 5. These values were calculated by weighing the corresponding D H B and following its quantitative cleavage using a Clark-type oxygen electrode. Table 5. Molar extinction coefficients of H O P D A s used in this study D H B Corresponding H O P D A Wavelength (nm) Extinction coefficient at pH 7.0 (mM-'cm- ' r Extinction coefficient at p H 6.5 ( m M ' c n f ' ) a D H B H O P D A 433 11.3 4.9 2'-Cl D H B 8-C1 H O P D A 392 28.1 N D 3'-Cl D H B 9-C1 H O P D A 436 17.5 N D 4-C1 D H B 3-C1 H O P D A 430 37.1 29.9 5-C1 D H B 4-C1 H O P D A 410 20.0 19.4 6-C1 D H B 5-C1 H O P D A 400 N D * 23.5 3',5'-diCl D H B 9,11-diCl H O P D A 438 19.6 17.9 5,3'-diCl D H B 4,9-diCl H O P D A 414 20.9 N D 2 ' ,6 ' -diCl D H B 8,12-d iClHOPDA 392 36.5 N D 4,3',5'-triCl D H B 3,9,11-triCl H O P D A 438 36.2 35.6 "Extinction coefficients were determined at 25° C using freshly generated H O P D A s and either 100 m M potassium phosphate buffer, p H 7.0 or 50 m M potassium phosphate buffer, pH 6.5. 6 N D , not determined. Figure 11. Molecular numbering of D H B and corresponding H O P D A . 3.1.3 Reactivity of D H B D L B 4 0 0 , D H B D P 6 - I , D H B D P 6 - I I I and T H B D R W I with chlorinated D H B Spectrophotometric assays demonstrated that each of the four type-I extradiol dioxygenases investigated in this study detectably cleaved each of the tested chlorinated DHBs . However, none of the four could cleave 3,4-DHB, and each enzyme had a distinct specificity for DHBs (see ka/p, Table 6). Indeed, among the enzymes, the specificity for a particular D H B differed by up to 3 orders of magnitude. Nevertheless, each enzyme was able to utilize at least one of the chlorinated D H B s as a better substrate than unchlorinated D H B . Inspection of the data did not reveal a consistent way to predict the reactivity of the di- and tri-Cl D H B s from those of the mono-Cl DHBs . For example, the reactivity of DHBDp6-I, DHBDp6-III and THBDRWI with 3 ' -Cl D H B correlated well their reactivity for 3 ' ,5 '-diCl D H B (i.e., the effect of the chloro substituents on the steady-state kinetic parameters appeared to be additive). However, this was not the case for DHBDLB40O- This isozyme cleaved 3' ,5 '-diCl D H B 7.7 times more specifically than D H B while 3 ' -C l D H B was a poorer substrate than D H B . Similarly, the presence of a 4-C1 substituent had a larger impact on the suitability of D H B as a substrate for DHBD L B4oo than on 3' ,5 '-diCl D H B . One striking feature of the data is that the A : ^ of the enzymes for di- and tri-Cl D H B s was almost always significantly lower than for D H B or mono-C l DHBs . This was true regardless of the suitability of the di- or tri-Cl D H B as a substrate The suitability of chlorinated DHBs as substrates for the dioxygenases can also be evaluated from the partition ratios. The latter is a measure of the average number of D H B molecules cleaved per enzyme molecule prior to oxidative inactivation of the latter. In principle, an enzyme should have a higher partition ratio for substrates that it has evolved to transform. As with the specificity constants, no consistent trend is immediately evident in the partition ratios or susceptibility to inactivation (j"pp' I KaJ%): each enzyme had a distinct pattern of susceptibility to inactivation by the different DHBs . However, the partition ratios of three of the enzymes (DHBD L B4oo, DHBDp 6 - I and THBDRWI ) were highest for D H B (Table 6). Although DHBDp6-III generally cleaved DHBs more slowly than the other three enzymes, this enzyme seemed best suited to the degradation of PCBs. More particularly, this enzyme showed a greater specificity for most of the tested chlorinated D H B s than for unchlorinated D H B . Most strikingly, the apparent specificity of DHBD P 6-III for 4,3'5'-triCl D H B was 28 times higher than for D H B . Similarly, the partition coefficients of the enzyme for many chlorinated D H B s were comparable to that for D H B . THBDRWI had a good specificity for 2 ' -C l D H B and had a relatively high partition coefficient in the presence of this substrate. This is consistent with the enzyme's physiological role, the cleavage of 2 ' -OH D H B in the degradation of dibenzofuran. Moreover, the specificity constant of THBDRWI for several chlorinated D H B was either very similar to or higher than that of unchlorinated D H B (3',5'-diCl D H B > 3 ' -Cl D H B > D H B ~ 2 ' -C l D H B ) . It was therefore somewhat surprising that among the tested enzymes, THBDRWI cleaved 2 ' ,6 ' -diCl D H B the least efficiently and had the lowest partition coefficient in the presence of this compound. Interestingly, no substrate inhibition was observed with any chlorinated DHBs even at a concentration of 3300 times Kapp in the case of 2'-CI D H B . Some substrate inhibition was observed in the presence of D H B , as reported by Happe et al. (1993), but the could not be reliably evaluated from the current data because experiments could not be performed at high enough substrate concentrations (Figure 12A). Finally, the enzyme's Km0^ in the presence of D H B was 0.8 ± 0.1 m M (Figure 12B), which is comparable to those reported for DHBDLB40o (1.2 ± 0.2 mM) and D H B D P 6 - I (0.25 ± 0.02 mM). 80 0 4 1 1 1 1 , , , 0 10 20 30 40 50 60 70 [ D H B ] O i M ) 200 [Oxygen] ( u M ) Figure 12. Steady-state cleavage of D H B by THBDRWI - A , The dependence of initial velocity on the concentration of D H B in air-saturated buffer. The line represents a best fit of the Michaelis-Menten equation to the data. The fitted parameters are Kapp = 2.8 ± 0.4 u M and V = 64 ± 2 uM/min. B , The dependence of initial velocity on the concentration of O2 using 100 u M D H B . The line represents a best fit of the Michaelis-Menten equation to the data. The fitted parameters are = 830 ± 1 0 0 u M and V = 200 ± 20 uM/min. A l l experiments were performed using 100 m M phosphate buffer, p H 7.0 at 25 °C and either 1.10 x 10"8 M (A) or 0.89 x 10"8 M (B) THBDRWI - Initial velocities obtained on different days were normalized according to the amount of enzyme used in the assay. Table 6. Apparent steady-state kinetic parameters and inactivation parameters of DHBDLB4oo, DHBDP6-I , DHBD P6-III and THBDRWI for chlorinated DHBs . Enzyme Substrate v-app ^mA jy-app h-aPP Kcal * 7 Partition Ratio • app Ji ;<>PP , V-OPP J3 IJ^mA ( U M ) (mM) (sec'1) (x 106 M V ) (x 10"3 sec'1) (x 103 M V ) DHBDLB4UO DHB" 2'-Cl DHB" 3'-Cl DHB" 4-Cl DHB" 3',5'-diCl DHB 2'6'-diCl DHB" 4,3'5'-tri CI DHB 6.9 (0.7) 6.4(0.5) 58(5) 16.4(1.8) 0.62 (0.07) 0.007(0.001) 1.1 (0.27) 3.6(0.9) 2.3 (0.4) 0.40(0.05) 3.5(1.1) 0.100 (0.035) 270 (6) 4.9 (0.1) 305 (20) 60.6 (2.2) 184 (6) 0.036(0.001) 5.5 (0.3) 39 (3) 0.76 (0.06) 5.3 (0.2) 3.7(0.3) 300 (25) 5.1 (0.9) 5.1 (0.4) 113 400 (600) 1 660 (300) 42 000(14 000) 14 200(760) 59 200(500) 49.5(1.6) 670(90) 2.4 (0.1) 2.9(0.6) 7.3 (2.9) 4.3 (0.4) 2.0(0.36) 0.69 (0.01) 7.6(0.6) 0.35 (0.05) 0.45 (0.13) 0.13 (0.06) 0.26(0.05) 3.2(0.9) 99(16) 7.0(2.3) DHBDP6-I DHB" 2'-ClDHB" 3'-Cl DHB" 4- Cl DHB" 5- Cl DHB" 3',5'-diCl DHB 5,3'-diCl DHB 2'6'-diCl DHB" 4,3'5'-tri CI DHB 5.8(1.0) 0.22 (0.06) 1.9(0.3) 22.0 (2.6) 5.3 (0.6) 0.20 (0.02) 0.37 (0.03) 0.145 (0.014) 0.46 (0.046) 2.2 (0.7) 1.5(0.3) 2.6 (0.7) 29.9(3.5) 78(3) 2.00 (0.04) 27.0(0.8) 110(5) 7.2 (0.2) . 36.0(0.8) 4.9(0.07) 0.088 (0.004) 25.2 (0.8) 13.3(1.9) 9(2) 14.2(1.8) 4.9 (0.4) 1.4(0.1) 180(18) 13.3(0.9) 0.61 (0.09) 54.5 (4) 31 100(3 300) 1 650(80) 2 290(190) 27 800 (200) 660 (35) 8 330(1100) 2 620 (20) 49.9(1.2) 6150(150) 2.5 (0.4) I. 2 (0.1) II. 8(1.3) 3.9(0.2) 11.0 (0.8) 4.1 (0.2) 1.8 (0.2) 1.7(0.1) 4.1 (0.4) 0.43 (0.14) 5.5 (2.0) 6.2(1.7) 0.18(0.03) 2.1 (0.4) 20.5(3.1) 4.9(0.9) 11.7(1.6) 8.9(1.8) DHBDp6-III DHB" 2'-Cl DHB" 3'-Cl DHB" 4- Cl DHB" 5- Cl DHB" 3',5'-diCl DHB 5,3'-diCl DHB 2'6'-diClDHB" 4,3'5'-triClDHB 17.0(1.9) 10.0(1.0) 10.0(0.7) 1.8(0.2) 4.3 (0.6) 3.5 (0.5) 1.8(0.2) 3.9 (0.4) 0.070 (0.009) 1.1 (0.4) 17.0(0.6) 5.3(0.1) 14.2(0.3) 4.0(0.1) 6.6 (0.3) 6.9 (0.2) 3.6(0.1) 0.069 (0.002) 1.9(0.05) 1.0(0.1) 0.50 (0.04) 1.40 (0.08) 2.2 (0.2) 1.5(0.2) 2.0 (0.2) 2.0 (0.2) 0.017 (0.001) 28 (3) 900 (60) 1990(160) 870(70) 260 (40) 100 (20) 560 (60) 330 (40) 15.0(0.3) 750 (150) 19.0 (2.0) 2.7(0.3) 16.3 (1.7) 15.5 (3.0) 68.0(17.1) 11.3 (0.6) 9.4 (0.3) 5.4 (0.2) 2.5 (0.5) . 1.1 (0.2) 0.25 (0.05) 1.6(0.3) 8.6 (2.6) 15.8(6.2) 3.2 (0.6) 5.2(0.7) . 1.4 (0.1) 36(12) THBDRWI D H B 2'-Cl DHB 3'-Cl DHB 4- Cl DHB 5- Cl DHB 3',5'-diCl DHB 5,3'-diClDHB 2'6'-diCl DHB 4,3'5'-tri CI DHB 2.80 (0.35) 0.11 (0.01) 0.56 (0.06) 5.6(0.8) 0.17(0.01) 0.120 (0.003) 0.046 (0.016) 0.150(0.006) 0.13 (0.02) c 97.5 (2.6) 3.1 (0.1) 30.5 (0.9) 2.9(0.1) 5.0(0.1) 17.5(0.1) 3.7(0.7) 0.00090(0.00002) 0.190(0.005) 35.0(7.5) 29.0(3.5) 55.0(7.5) 0.52 (0.09) 30.0 (2.4) 150(5) 80 (30) 0.006 (0.002) 1.5(0.2) 59300(1800) 9200 (300) 8800 (1100) 6800 (240) 5500 (150) 960(60) 710(200) 9.4 (0.2) 74 (3) 2.30 (0.01) 0.310(0.001) 5.00 (0.05) 0.670 (0.001) 1.33 (0.01) 18(1) 5.2(0.5) 0.120(0.004) 2.60 (0.03) 0.80 (0.06) 3.1 (0.4) 8.9(1.8) 0.11 (0.02) 7.8(0.9) 160(13) 110(40) 2.4 (0.2) 20 (3) The meaning of rate parameters is summarized in Scheme I (Section 1.5). Values in parentheses represent standard errors. a Taken from (Vaillancourt et al. 2003). b— signifies that no inhibition was observed. cKlh estimated at 1 m M . 3.2 Biological selection and screening host cells (SHCs) Six biological selection and screening host strains (SHCs) were developed during the course of this PhD work. Each is based on the detection of benzoate, an end product resulting from the transformation of biphenyl and some PCBs by the upper bph pathway (Figure 2). The SHCs were engineered by inserting two elements into the chromosome of E. coli or P. putida: (1) a copy of xylS and (2) a reporter gene under the control of Pm, the XylS-activated promoter. X y l S is a transcription activator of the AraC family that regulates the expression of the meta-T O L pathway of P. putida mt-2 (Martin and Rosner 2001). X y l S triggers transcription from the Pm promoter in the presence of benzoate effectors (Ramos et al. 1997). It is therefore reasonable to expect that placing an antibiotic resistance under the control of the Pm promoter would couple the transformation of biphenyl to the viability of the cell in the presence of the corresponding antibiotic. Such a system could be adapted to select PCB-degrading enzymes, including engineered D H B D s and H O P D A hydrolases (Figure 13). The SHC1 and SHC2 systems were constructed in E. coli by placing the cat gene, f I f 1 i / / CZ> 1 I xylS Pm antibiotic resistance gene Figure 13. Principles used to design the selection-based SHCs for the directed evolution of PCB-degrading enzymes. encoding a resistance against chloramphenicol, under the control of the Pm promoter. SHC4 and SHC7 were constructed in E. coli and P. putida, respectively, by using the tetracycline resistance-encoding tetC gene as the benzoate-inducible selection marker. Finally, two biological screening host cells, SHC11 in P. putida and SHC12 in E. coli, were obtained by employing the luxCDABE cassette as a reporter. 3.2.1 E. coli S H C l : towards a benzoate-inducible chloramphenicol resistance system The cat gene was originally chosen as a selection marker because C A T confers a chloramphenicol resistance that is proportional to its expression level in the cell (Brosius 1984). Thus, cells producing more benzoate would have a higher intracellular level of C A T , resulting in a higher growth rate in the presence of chloramphenicol. In principle, this characteristic would enable the rank-ordering of colonies based on activity of PCB-transforming enzymes. The cat gene of pKK232-8 was cloned into pUC18Not to facilitate its subsequent cloning into p C N B l , a mini-Tn5 delivery vector. The resulting plasmid, p C N B l C A T , contained xylS under the control of the constitutive Ps2 promoter (de Lorenzo et al. 1993b) and the cat gene under the control of the Pm promoter. E. coli S17.lA.pir was transformed with p C N B l C A T and used for preliminary characterization of the system. The cells were grown on L B solid medium containing chloramphenicol (25 ug/ml) and a range of concentrations of benzoate (2-100 uM). As summarized in Table 7, 2 u M benzoate was sufficient to induce resistance against chloramphenicol and colony size was proportional to the benzoate concentration. However, a number of colonies, representing up to 5 % of the original inoculum, appeared on the selection medium after 18 hours of incubation, in the absence of benzoate. This phenomenon is referred to herein as background growth. Table 7. Regulation of chloramphenicol resistance by benzoate in E. coli S17.lA.pir ( p C N B l C A T ) . Chloramphenicol (ug/ml) Benzoate concentration (uM) Colony diameter (mm) 0 0 0.75-1.00 25 0 -25 2 0.15-0.20 25 5 0.20-0.30 25 10 0.30 25 25 0.30 25 50 0.40-0.50 25 100 0.50-0.75 Cultures were grown for 18 hours at 37°C on L B solid medium containing 100 ug/m] ampicillin. Conjugation experiments were carried out between the donor strain, E. coli S17.lA.pir ( p C N B l C A T ) , and the recipient strain, E. coli CC118Rif. The spontaneous rifampicin resistant derivative of E. coli C C l 18 was used to facilitate the selection of the exconjugants. Clones characterized by rifampicin resistance, kanamycin resistance and ampicillin sensitivity were identified as exconjugants that had the mini-transposon inserted into their chromosome. The frequency of transposition of the mini-Tn5 construct into E. coli CC118Rif was 8.8 x 10"4 (-35 positive clones per 4 x 105 cells). A n ex-conjugant was chosen and designated E. coli S H C l . This strain, which contained xylS, Pm and cat on a single module, was not resistant to chloramphenicol in the presence of benzoate. The lack of cat expression could be due to low levels of X y l S in the cell. Accordingly, E. coli S H C l was transformed with pVLT43, which harbours the xylS gene under the control of the IPTG-inducible Plac promoter. E. coli S H C l (pVLT43) constitutively expressed C A T and was resistant to chloramphenicol (25 ug/ml), even in the absence of benzoate and IPTG. It is known that high levels of X y l S induce transcription from Pm even in the absence of benzoate (Ramos et al. 1997). Overall, these experiments suggested that E. coli S H C l is o f limited use as a selection host due to the intracellular levels of X y l S . 3.2.2 SHC2: a bimodular benzoate-inducible chloramphenicol resistance system To overcome the limitation of E. coli S H C l , E. coli S H C 2 was designed such that the expression of X y l S would be regulated by an independently inducible promoter. The elements introduced into E. coli S H C 2 were similar to those introduced into E. coli S H C l but were divided into two complementary modules. One module carries the cat gene under the control of the Pm promoter and the second, the xylS gene under the control of the IPTG-inducible promoter (Figure 14). The two constructions were successively introduced into the chromosome of E. coli C C l 18 (rif) following the same strategy used for the E. coli S H C l system. E. coli S H C 2 grew on solid L B medium containing 25 u.g/ml of chloramphenicol, 25 u M I P T G and benzoate concentrations ranging from 2 u.M to 5 m M . Moreover, colony size was proportional to benzoate concentration over a range from 2 to 100 u M . The average size of a colony grown on L B solid medium containing 25 u.g/ml chloramphenicol, 25 u M IPTG and 100 u M benzoate was 80 % that of colonies grown on L B solid medium without selection. Finally, no growth was observed after 18 hours of incubation in the absence of benzoate. Figure 14. The two mini-Tn5 modules that were stably inserted into the chromosome of E. coli C C l 18Rif to create E. coli S H C 2 . Additional details are provided in Table 2. E. coli SHC2 was transformed with plasmid p M A D D B , which contained the dbfB and bphD genes under the control of the Pbad promoter. E. coli SHC2 ( p M A D B D ) was used to test the response of the system to benzoate produced in vivo from the transformation of D H B by the tandemly expressed THBDRWI and H O P D A hydrolase. As summarized in Table 8, no growth was observed in the presence of chloramphenicol and D H B . Furthermore, colonies grown in the presence of benzoate and D H B were 75% smaller than those grown in the presence of benzoate only. Finally, incubation times of more than 18 hours resulted in the background growth of E. coli SHC2 ( p M A D D B ) on the selection medium in the absence of benzoate. A n experiment in which the concentrations of benzoate and D H B were varied from 0 to 100 u M and 0 to 400 u M , respectively, yielded essentially identical results (not shown). Table 8. E. coli SHC2 ( p M A D D B ) growth in the presence of chloramphenicol, benzoate and D H B . Selection reagent Growth Chloramphenicol (u.g/ml) benzoate (uM) D H B (uM) 0 0 0 + 25 0 0 -25 100 0 + 25 100 200 + b 25 0 200 Cultures grown for 18 hours at 37 °C on L B solid medium containing 100 ug/ml ampicillin, 25 u M IPTG, 0.2 without D H B . % arabinose. bsize of the colonies were ~ 75% smaller than the colonies grown To investigate whether the levels of expression of THBDRWI and H O P D A hydrolase were sufficient for benzoate production from D H B degradation, E. coli SHC2 ( p M A D B D ) was grown on L B solid medium containing 100 ug/ml ampicillin, 25 ug/ml chloramphenicol, 25 u M IPTG, 0.2% arabinose and 100 u M benzoate. The colonies were resuspended in potassium phosphate buffer pH 7.0 (1= 0.1 M) , the cells were broken open using a French Press, and the 58 enzymatic activities were assayed in the raw extracts. The activities of THBDRWI and H O P D A hydrolase were approximately 60 and 14 U/ml of E. coli SHC2 cytoplasm, respectively. 3.2.3 E. coli SHC4 and P. putida SHC7: bimodular benzoate-inducible tetracycline resistance Due to the limitations of the E. coli SHC2, an additional system, based on a different antibiotic resistance, was constructed. E. coli SHC4 {Ptrc-xylS, Pm-tetC) was engineering in E. coli CC118Rif essentially as described for E. coli SHC2 except that the cat gene was replaced by the tetracycline resistance gene (tetC). E. coli SHC4 grew on L B solid medium containing 7.5 ug/ml tetracycline, 25 u M IPTG, and benzoate concentrations ranging from 10 to 100 u M . E. coli SHC4 was transformed with p M A D B D and tested for the in vivo formation of benzoate, essentially as described for E. coli SHC2. The presence of 25-200 u M D H B failed to induce tetracycline resistance. Furthermore, incubation of cells for 18 hours resulted in background growth on the selection medium in the absence of benzoate. D H B also appeared to be toxic to the cells as the size of the colonies was inversely proportional to the concentration of D H B in the medium (results not shown). In an attempt to overcome the limitations of E. coli SHC4 caused by D H B toxicity, the same system was constructed in P. putida CL01 , yielding P. putida SHC7 (P,rc-xylS, Pm-tetC). P. putida SHC7 was able to grow in the presence of tetracycline (15 ug/ml) and benzoate concentrations ranging from 10 to 100 u M (Table 9). Remarkably, no background growth was observed in the absence of benzoate after 66 hours of incubation at 30 °C. Moreover, the colonies grown in the presence of 200 u M D H B were similar in size to those grown without D H B . Table 9. Growth of P. putida SHC7 in the presence of, benzoate and D H B . Selection reagent Growth Benzoate (uM) D H B (uM) 20h 42h 66h 0 0 -5 0 10 0 + 25 0 + + 50 0 + . + + 100 0 + + + 100 200 + + + The cultures were incubated at 30 °C on L B solid medium containing 15 ug/ml tetracycline. P. putida SHC7 was transformed with pUCP24BD, an E. coli/Pseudomonas shuttle vector containing dbfB and bphD under the control of the IPTG-inducible P i a c promoter. P. putida SHC7 (pUCP24BD) was grown on L B solid medium in the presence of 15 ug/ml tetracycline, 25 u M IPTG and 0 to 200 u M D H B . The same medium containing 100 u M of benzoate was used as a positive control. As observed with E. coli SHC2 and E. coli SHC4, the presence of D H B failed to induce the growth of P. putida SHC7 in the presence of tetracycline. 3.2.4 P. putida SHC11 and E. coli SHC12: luminescence-based biological screening host cells Due to the problems encountered in establishing a biological selection system, screening systems were designed exploiting many of the elements used in constructing the selection systems except that the antibiotic resistance gene was substituted with the luxCDABE cassette, which produces light. More specifically, P. putida SHC 11 (Ptrc-xylS, Pm-luxCDABE) was similar to E. coli SHC2 but contained the lux genes under control of the Pm promoter. Similarly, E. coli SHC 12 (Ptrc-xylS, Pm-luxCDABE) was similar to E. coli SHC2 but contained the lux genes under control of the Pm promoter. P. putida SHC11 and E. coli SHC 12 were evaluated by following the production of luminescence in aliquots of mid-log phase cells in the presence of 0 to 100 u M benzoate using a microtiter plate. A s shown in Figure 15A, the induction o f luminescence in P. putida S H C 11 was proportional to the benzoate concentration in the medium (Figure 15A). Interestingly, the luminescence emitted by P. putida S H C 11 increased exponentially for 3 hours in all cultures containing benzoate but then dropped during the fourth hour. Time (minutes) 1.4 -, Time (minutes) Figure 15. Induction o f luminescence by benzoate in P. putida S H C 11 and E. coli S H C 12. Luminescence was monitored for 4 hours at 10 minutes intervals using a 96-well microtiter plate. A ) Wells contained 200 ul of a mid-log phase culture of P. putida SHC11 ( O D 6 0 0 = 0-3) and from 5 to 100 u M benzoate. Plates were incubated at 30 °C. B) Wells contained 200 ul of a mid-log phase culture of E. coli S H C 12 (OD600 =0.3) and from 5 to 100 u M benzoate. Plates were incubated at 37 °C. Cultures incubated in the absence of benzoate served as reference. Assays were performed in triplicate. Error bars represent the standard deviation. R L U , relative light units. Additional details are provided in section 2.5. The production of luminescence in E. coli SHC 12 was also proportional to the concentration of benzoate in the medium (Figure 15B). However, the kinetics of light production in E. coli SHC 12 seemed to be slower than in P. putida SHC11. Thus, the intensity of luminescence in E. coli SHC 12 was lower than in P. putida SHC11. For example, after 3 hours of incubation in the presence of 100 u M benzoate, the luminescence produced by E. coli SHC 12 was approximately 5 times lower than by P. putida SHC 11. Moreover, the luminescence continued to increase in a linear fashion until the end of the experiment in E. coli SHC12, in contrast to peaking after 3 hours as observed in P. putida SHC11. P. putida SHC 11 and E. coli SHC 12 were transformed with pUC24BD and p M A D B D , respectively, to evaluate the induction of luminescence from the in vivo formation of benzoate from D H B . Interestingly, the presence of D H B in the medium caused a significant decrease in luminescence. Aliquots of mid-log phase cultures of E. coli SHC 12 ( p M A D B D ) were incubated in the presence of different concentrations of D H B ranging from 0 to 500 u M . The background luminescence of the cultures containing D H B was lower than the ones without D H B (Figure 16). 20 100 DHB concentration (uM) 500 Figure 16. Luminescence of E. coli SHC 12 ( p M A D B D ) in the presence of D H B . Assays were performed in triplicates. Error bars represent standard deviation to the values. R L U , relative light units. Moreover, the decrease in luminescence appeared to be exponentially proportional to the concentration of D H B in the medium (results not shown). The experiment was repeated with P. putida S H C l 1, with a similar outcome (results not shown). The induction of luminescence by the in vivo formation of benzoate from D H B was therefore not observed in either system. In addition, the overall results of were inconclusive. Efforts were redirected to develop a high-throughput colorimetric screen for the directed evolution of extradiol dioxygenases (see section 3.2.5). 3.2.5 Colony screen A high-throughput colorimetric screen was designed to identify extradiol dioxygenase variants with improved specificity for particular catechols (see section 2.8). The screen was validated by improving the 3,4-DHB-cleavage activity of DoxG. Accordingly, libraries of mutated DoxG were plated on solid medium and sprayed with 5 m M 3,4-DHB. Colonies containing a 3,4-DHB-cleavage activity developed the yellow colour characteristic of the meta-cleavage products (enolate anion). Images were captured through a 400 nm optical filter 2 minutes after exposure to 3,4-DHB and processed to extract the blue channel (Figure 17A-B). In the resulting image, the colonies producing yellow products appeared as dark spots against a light background (Figure 17B). The intensity of these spots could be quantified using ImageQuant 5.2 (Figure 17C). Analysis of the processed images obtained using a library of randomly mutated DoxG (section 3.3.1.2) revealed that a small number of colonies (< 0.5%) developed more intense colour than colonies containing the wild-type enzyme. Attempts to follow the development of the yellow colour with time (30 s intervals) were not successful. Nevertheless, the colonies showing the highest pixel density were picked and grown in 96 deep-well plates in 1 ml L B medium containing 0.5 m M IPTG and 100 mg/1 ampicillin. Filtered image B 1 2 3 4 colony Figure 17. Quantification of ring-cleavage activity in plated colonies using digital imaging. A ) Colonies of E. coli Nova Blue DE3 transformed with pT7-7 containing a library of randomly mutated doxG were grown to a density of 10 cfu/cm 2 and sprayed with a 5 m M solution of 3,4-D H B . B) Images were captured through a blue filter (max. transmission at 450 nm) and processed to extract the blue channel. Active clones appear as dark spots. C) The pixel density (pixel/cm 2) corresponding to each active clones was determined using the ImageQuant 5.2 software (Amersham Biosciences). 3.3 Directed evolution of D o x G The extradiol dioxygenases presented in section 3.2 all shared the common inability to cleave 3,4-DHBs. In contrast, D o x G , which catalyzes the extradiol cleavage of 1,2-DHN in the catabolism of naphthalene, has a low specificity for 3 ,4-DHB (Barriault et al. 1998). Consequently, a directed evolution approach was used to improve the 3,4-DHB activity of D o x G . 3.3.1 Library generation and analysis 3.3.1.1 Error-prone PCR A library of DoxG variants was generated by random mutagenesis using error-prone PCR. Sequence analysis of 6 randomly selected clones indicated that the mutation rate was 0.7% at the nucleotide level, corresponding to an average of 2 amino acid changes per enzyme variant. This analysis further indicated that the rates of transition and transversion mutation were equivalent. Libraries of over 2 x 105 clones were routinely obtained from a single ligation reaction containing 150 ng of D N A . A library of 25,000 clones was screened for improved 3,4-DHB cleavage activity. When sprayed with 5 m M 3,4-DHB, -20% of the plated colonies developed a yellow coloration. Ninety colonies were selected for further screening using a 96-well microplate. In the presence of 0.1 m M 3,4-DHB, seven DoxG variants showed a 1.6- to 5-fold increase in cleavage activity versus the wild-type enzyme (Table 10). To confirm that activity increases were not due to differences in expression levels, the activity of the variants was also assayed using D H B . Those variants possessing the largest change in activity ratio were further characterized. Analyses of cell extracts of these clones using Sypro Ruby-stained S D S - P A G E revealed that the overall difference in expression levels of the variants was less than 20% (results not shown). Mapping the mutations onto the structure of the DoxG:3,4-DHB complex revealed that all but one variant had at least one substitution in a residue that contributes to the substrate-binding pocket (Figure 18). The exception was variant B8 which contains the same two substitutions as IP33 (L242S and P267L) in addition to the Q101R substitution. Comparison of the B8 and IP33 suggests that the Q101R mutation increased substrate preference for 3,4-DHB relative to D H B by 12-fold (Table 10). Within a single protomer, this residue is 30 A away from the enzyme's active site iron yet had a significant effect on the substrate preference of the enzyme. Table 10. Relative activities of selected DoxG variants obtained by error-prone PCR. Relative Increase in Variant Mutations 3,4-DHB activity 3 , 4 - D H B activity ratio (fold) (variant/WT) ^ D H B activity , WT None 1 1 IP27 I154F 4.6 6.6 IP33 L242S 5.1 1 P267L IP36 D21E 1.8 N D a V290A IP37 I154F 4 12.1 L182S I265L K300I IP38 H59L 1.6 N D K165R L 2 5 3 M B l L242S 3.8 43 P267L S191T B8 L242S 3.5 12 P267L Q101R Assays were performed using 100 u M substrate and 100 m M phosphate buffer, pH 7.0 at 25°C. Activities determined using cell raw extracts and normalized according to expression levels of DoxG variants (see section 2.9). a Not determined. 3.3.1.2 Saturation mutagenesis For any given residue, an average of 5.7 amino acid substitutions should be accessible by random mutagenesis (Miyazaki and Arnold). To increase the number of substitutions sampled at key positions, four residues were subject to saturation mutagenesis: Ilel54, Leul90, Serl91 and Leu242. These residues are located within 14 A of the enzyme's active site iron, and the random mutagenesis results showed that their substitution had a large effect on the enzyme's activity. To Figure 18. The structure of D o x G bound to 3 ,4-DHB. The green sphere represents the active site ferrous ion. The bound substrate is shown in yellow. A ) Point mutations observed during random mutagenesis are shown in red. B) Crystal structure showing the interface of two doxG monomers. The Q101R mutation is situated 4 A away from Arginine 179, located next to Tyrosine 178 that shifts upon substrate binding. Figure made using P y M O L v.0.98 (Delano 2005) and P D B coordinates of the DoxG:3 ,4 -DHB binary complex (Neau 2004) manage the size of the experiment, the residues were mutated pairwise: 154 and 242 in one trial; and 190 and 191 in the second. The two resulting libraries consisted of variants containing all possible 20 amino acids at each target position. This strategy allowed us to explore greater than >99.9% of the possible diversity while screening less than 10,000 clones. A n initial agar plate-based screening of -25,000 clones from each library followed by microplate-based screening of promising clones identified numerous variants displaying 2- to 10-fold increases in 3 ,4-DHB cleavage rates using 100 u M 3,4-DHB (Table 11). A l l variants contained mutations involving hydrophobic residues lining the substrate-binding pocket of the enzyme. 3.3.1.3 DNA shuffling The 6 best clones obtained by saturation mutagenesis, with the addition of variants IP27 and IP33, were used in a DNA-shuffling experiment. Sequencing of 5 randomly picked clones indicated that the shuffling efficacy was 2-3 recombination events per clone. A library of -25,000 clones was screened for improved 3,4-DHB degrading activity. The most active variant, DoxG S M A2, contained three mutations: L190M, S191W and L242S (Figure 19). The sequence indicates that this variant resulted from the recombination of three of the six parental variants used in the shuffling experiments (IP33, SB6 and SCI) and that one of the recombination events occurred between adjacent codons that were mutated with respect to the wild-type doxG gene. Table 11. Relative activities of selected DoxG variants obtained by saturation mutagenesis. Variant Mutations Relative 3,4-DHB activity (variant/WT) Increase in 3 , 4 - D H B activity A D H B activity ratio (fold) WT None 1 SA8 L242V 1.7 10 SA12 L242T 2.6 63 SB1 L242G 6.4 18.1 SB2 I154W 9 12.3 SB4 I154W 8.8 12.1 SB6 S191W 3.3 80.2 L242K SCI L 1 9 0 M 9.9 15.3 S191Y L242I SG12 L190Y 4.7 20.9 L242I Assays were performed using 100 u M substrate and 100 m M phosphate buffer, p H 7.0 at 25°C. Activities determined using cell raw extracts and normalized for expression levels of DoxG variants (see section 2.9). Figure 19. The structure of D o x G bound to 3,4-DHB showing the D O X G S M M variant mutations. Tracings o f the N - and C-terminal domains are coloured slate and gray, respectively. The green sphere represents the active site ferrous ion. Residues 190, 191 and 242 are shown in straw. The bound substrate is represented in yellow. A ) Mutated residues on W T D o x G structure. B) Surface model of the DOXGSMA2 substrate pocket. Figure made using P y M O L v.0.98 (Delano 2005). The images are based on the crystal structure of the DoxG:3 ,4 -DHB binary complex (Neau 2004). 3.3.2 Characterization of W T and variant DoxG 3.3.2.1 Purification of DoxG and DOXGSMA2 DOXGSMA2 was purified anaerobically to apparent homogeneity as summarized in Table 12. Purified DOXGSMA2 contained greater than 96% of its complement of iron and had a specific activity for D H B of 1.9 U/mg using D H B as substrate. Preparations of D o x G were of a quality similar to that obtained previously (Neau 2004). Table 12. Purification details of DOXGSMA2-c ™ J -f t- + Total protein Total activity Specific Activity Yie ld Enzyme and purification step , F . n i . J m i \ ro/\ J v y (mg) (U) (U/mg) Raw extract 730 210 0.29 100 MonoQ 100 130 1.30 59 Superdex200 80 150 1.90 70 Activity units are defined in section 2.11.1. 3.3.2.2 Steady-state kinetic analysis Steady-state kinetic studies were performed to investigate the combined effect of the three mutations on the specificity of the enzyme. Enzymatic assays were performed in air-saturated buffer using 1,2-DHN, D H B and 3,4-DHB as substrates. The data were fit to the Michaelis equation (Table 13). The variant's k"pp for 3,4-DHB was 2.4-times higher than that of the wild-type (WT) DoxG. However, the Kapp value of the variant for 3,4-DHB was much lower, resulting in an apparent specificity constant almost three orders of magnitude greater than that of WT enzyme. The specificities of the variant for 1,2-DHN and D H B were also changed with respect to the WT enzyme, but to much lesser extents (6.7- and 0.5-fold, respectively). Notably, for each substrate, the k"™ values of the two enzymes varied by less than a factor of three. Table 13. Apparent steady-state kinetic parameters of DoxG and DOXGSMA2-Variant Substrate l-app Kcal Kapp m U"PP KA G*M) (103 M^s" 1) W T 1,2-DHN 9.6 (0.8) 120(20) 77 (7) W T D H B 1.8 (0.04) 2.6 (0.3) 680 (60) WT 3,4-DHB 1.6 (0.07) 600(100) 2.6 (0.3) S M A 2 1,2-DHN 6.1 (0.3) 11.8(1.9) 520 (70) S M A 2 D H B 0.70 (0.03) 2.0 (0.4) 350 (60) S M A 2 3,4-DHB 3.8 (0.1) 1.9(0.3) 2000 (300) Assays were performed using 100 m M phosphate buffer, p H 7.0 at 25°C. The values in parentheses represent standard errors. 3.3.2.3 Inverse analysis of DOXGSMA2 mutations As discussed below, the L242S substitution occurs on one side of the substrate-binding pocket whereas the L 1 9 0 M and S191W substitutions occur on the other. To evaluate the effect of these substitutions on the substrate preference, two variants of DoxG were prepared which contained the L242S and L190M/S191 substitutions, respectively. The relative specific activity of the four variants towards 1,2-DHN, D H B and 3,4-DHB are summarized in Table 14. Although DOXGSMA2, containing all three mutations, possessed a significantly higher activity towards 3,4-DHB, the two sets of substitutions had opposite effects on the enzyme's 3,4-DHB cleavage activity when studied in isolation. Thus, while the 3,4-DHB cleavage activity of the L242S variant was 2.5-fold higher than that of the wild-type enzyme, that of the L190M/S191W variant was 3-fold lower. By contrast, both variants were less active towards 1,2-DHN and Table 14. Apparent relative activity of DoxG variants against 1,2-DHN, D H B and 3,4-DHB. Variant Substrate W T D o x G L242S L190M/S191W DoxG S M A2 1,2-DHN 100* 7.3 37 7.8 D H B 100 7.1 30 24 3,4-DHB 100 250 38 1020 Assays were performed using 100 u M substrate and 100 m M phosphate buffer, pH 7.0 at 25°C. Activities determined using cell raw extracts and normalized for expression levels of DoxG variants. "Activities are expressed as a percentage relative to that of W T DoxG. D H B . Inverse analysis using DOXGSMA2 as a reference (Mildvan 2004) suggests that the effects of the two sets of substitutions are antagonistic for 1,2-DHN- and DHB-cleavage activities and anti-cooperative for 3,4-DHB cleavage activities (Figure 20). 3.3.2.4 H P L C analysis of 3 ,4-DHB cleavage products The cleavage products of 3,4-DHB formed by W T DoxG and DOXGSMA2 were analyzed by H P L C . The cleavage of 3,4-DHB catalyzed by WT DoxG resulted in the formation of two products, 4-phenyl H O D A and 5-phenyl H O D A , in a 2.8 to 1 ratio, consistent with previous results (Barriault et al. 1998). The DoxGsMA2-catalyzed cleavage of 3,4-DHB yielded the same two products, but in an 8 to 1 ratio. Analysis of the cleavage products formed by the L242S and L190M/S191W variants indicated that the L242S substitution was essentially entirely responsible for the difference in product formation ratio observed in the S M A 2 variant. 0.5 | 0 u CO 0 > -0.5 JO o> o -2.5 1 — -WT L242 1 WT L242 L190 L190 S191 S191 1190 S191 L242 WT 1,2-DHN 2,3-DHB 3,4-DHB D0XGSMA2 Figure 20. Inverse analysis of the effect of DOXGSMA2 substitutions on D o x G activity. Two sets of substitutions were investigated: L242S and L190M/S191W. The graph plots the log of the relative activity of each variant versus D O X G S M M for each of three substrates Accordingly, the baseline (0) represents the relative activity of DOXGSMA2- The dotted bars plotted for W T D o x G represent what the relative activity o f the W T and DOXGSMA2 would be i f the effects of the two sets of substitutions was additive. 3.4 Transformation of P C B metabolites by B p h K 3.4.1 Expression and Purification of B p h K BphK was expressed to very high levels using E. coli GJ1158 and p T 7 B K l , constituting -50% of the soluble cellular protein (Figure 21). Relevant details of the purification of over 160 mg of BphK from 1 L of cell culture are presented in Table 15. The obtained BphK was >99% homogeneous as judged by S D S - P A G E (Figure 21) and had a specific activity 7.8 U/mg towards the reporter substrate C D N B . Table 15. Purification details of BphK expressed in E. coli GJ1158. Purification step Total protein Total activity Specific activity Yield (%) (mg) (U) (U/mg) Raw extract 338 1510 4.5 100 GSH-column eluate 169 1310 7.8 87 Figure 21. Analysis of purified BphK using denaturing gels. Lanes were loaded with (2) crude extract of E.coli GJ1158(pT7BKl), (3) 20 ug of column flow-through, and the eluted BphK samples: (4) 5 ug, (5) 10 ug, (6) 20 ug and (7) 50 ug. Lane 1 contains a molecular weight standard. The molecular weights of the proteins in the standard are indicated beside the gel. The gel contained 12% acrylamide and was stained with Coomassie Blue. 3.4.2 BphK-catalyzed transformation of chlorinated DHBs, HOPDAs and 4-CBA The ability of BphK to catalyze the transformation of a variety of P C B metabolites was tested by following the depletion of these compounds using analytical H P L C . In reaction mixtures containing up to 5 m M G S H and 2 m M BphK, the enzyme did not detectably transform any of the following DHBs when present at concentrations up to 2.5 m M : 4-C1 D H B , 5-C1 D H B and 6-C1 D H B . In contrast, BphK transformed 3-C1 H O P D A (50 u M in assay) , 5-C1 H O P D A (50 u M in assay) and 3,9,11-triCl H O P D A (75 u M in assay) with apparent specific activities of (3.60 ± 0.05) x 10"2 U/mg, (2.45 ± 0.05) x 10"2 U/mg and (0.22 ± 0.03) x 10"2, respectively. As 3-C1 H O P D A s are inhibitory P C B metabolites (see section 1.7), their transformation by BphK was studied in more depth, as described below. BphK did not detectably catalyze the transformation of either 4-C1 H O P D A or unchlorinated H O P D A . It was previously reported that BphK catalyzes the dechlorination of 4 - C B A (Gilmartin et al. 2003). When a solution containing 2.5 m M 4 - C B A and 5 m M G S H was incubated in the presence of 2 m M BphK, 50% of the 4 - C B A was dechlorinated after 2.5 h. This corresponds to an estimated enzymatic turnover number of 7 x 10"5 s"1. Under similar conditions, the specific activity of BphK for C D N B , a reporter substrate used in the characterization of GSTs, corresponded to an apparent &c at of 2.9 s"1. 3.4.3 Demonstration that BphK catalyzes the dechlorination of 3-C1 HOPDAs The reaction products of the BphK-catalyzed transformation of 3-CI H O P D A were analyzed using a chloride ion assay and H P L C . The former demonstrated that chloride was stoichiometrically released during GSH-dependent transformation of 3-C1 H O P D A (Figure 22). Moreover, two equivalents of G S H were utilized per molecule of 3-C1 H O P D A during the dechlorination reaction (not shown). Analysis of the reaction mixture by H P L C revealed a product with a retention time (tR = 16.1 min), and absorption spectrum (Figure 23) that corresponded exactly to those of unchlorinated H O P D A (tR of 3-C1 H O P D A = 15.3 min). Finally, when incubated in the presence of the H O P D A hydrolase, BphDLB400, the product was transformed to the two expected products, hydroxypentadienoate and benzoate. 20 Time (min.) Figure 22. Dechlorination of 3-C1 H O P D A by BphK. Transformation of 3-C1 H O P D A (A) coupled to the release of chloride ions (•). Reaction performed in duplicate using potassium phosphate, p H 6.5 (I = 0.05 M ) , 25 °C. Error bars represent the standard deviation. 200 250 300 350 wavelength (nm) 200 ' 250 ' ' 300 350 wavelength (nm) Figure 23. U V - V i s absorption spectra of (A) 3-C1 H O P D A , (B) H O P D A and (C) product of BphK-catalyzed transformation of 3-C1 H O P D A . Spectra were collected in a 50:50 mixture of methanol:water using a Waters 2996 photodiode array detector during H P L C analysis. 3.4.4 Steady-state kinetics with 3-C1 HOPDA, 3,9,11-triCl HOPDA and GSH 3.4.4.1 Steady state kinetics using fixed concentrations of GSH and HOPDAs To facilitate the steady-state kinetic characterization of the BphK-catalyzed transformation of 3-C1 H O P D A s , a continuous, spectrophotometric assay was developed. This assay was based on the differential absorption of chlorinated and non-chlorinated H O P D A s ( A s 3 C H = 21.5 m M ' W and AstnCH = 17.7 mAT'cm" 1 for 3-C1 H O P D A and 3,9,11-triCl H O P D A , respectively; I = 0.05 M potassium phosphate, pH 6.5) Experiments performed using 1 m M G S H revealed that the enzyme catalyzed the transformation of 3-C1-HOPDA (Figure 24) and 3,9,11-triCl H O P D A following simple Michaelis-Menten kinetics. The apparent specificity of BphK for 3-C1 H O P D A was (5.6 ± 0.9) x 103 NT's"1. That for 3,9,11-triCl H O P D A was -16 times lower. The enzyme's apparent Km values for 3-C1 H O P D A and 3,9,11-triCl H O P D A were (18 ± 2 ) and (44 ± 5) u M , respectively. Steady-state kinetics performed using fixed concentrations of H O P D A s revealed that the nature of the H O P D A strongly influenced the apparent Km for G S H . Thus, using 50 u M 3-C1 H O P D A , the apparent Km of BphK for G S H was 124 u M (Figure 24). In contrast, in the presence of 75 u M 3,9,11-triCl H O P D A , the apparent Km for G S H was 2.0 m M . 3.4.4.2 Steady state kinetics using varied concentrations of GSH and HOPDAs To investigate the steady-state mechanism of the BphK-catalyzed dechlorination of 3-C1 H O P D A s and to better estimate the rate parameters of this process, initial reaction rates were measured over a range of G S H and 3-C1 H O P D A s concentrations. Two sets of experiments were performed using (a) 5-50 u M 3-C1 H O P D A and 50-1000 u M G S H and (b) 10-50 u M 3,9,11-triCl H O P D A and 500-3500 u M G S H . A n equation describing a substituted-enzyme mechanism could not be fit to either data set. In contrast, an equation describing a ternary complex mechanism (Cornish-Bowden 1995) fit both data sets with random trends in the residual plots. This result was confirmed graphically by primary plots of [CI HOPDA]/v vs. [CI H O P D A ] which yielded a set of lines that intersected at a point on the negative side of the abscissa (Figure 25). Nevertheless, it was not possible to distinguish between a rapid-0 20 40 60 80 [3-CI H O P D A ] (MM) Figure 24. Steady-state transformation of 3-CI H O P D A by BphK. The curve represents a best fit of the Michaelis-Menten equation to the data using the least squares, dynamic weighting options of L E O N O R A (Cornish-Bowden 1995). The main panel depicts the dependence of initial velocity on the concentration of 3-CI H O P D A in the presence of 1 m M G S H . The fitted parameters are Kafa_H0PDA = 22 ± 2 uM and V= 0.41 ± 0.02 uM/min. The insert depicts the dependence of initial velocity on the concentration of G S H using 50 uM 3-C l H O P D A . The fitted parameters are KafGSH = 120 ± 20 uM and V= 0.32 ± 0.03 ^M/min. Experiments were performed using potassium phosphate, pH 6.5 (I = 0.05 M ) , 25 °C and either 5.5 x 10"8 M (main) or 4.3 x 10~8 M (inset) BphK. equilibrium random-order ternary-complex and a compulsory-order ternary-complex mechanism using the current data, as the two are kinetically equivalent (Cornish-Bowden 1995). The kinetic parameters obtained by fitting an equation describing a ternary complex mechanism to the data are provided in Table 16. The Km of BphK for 3-C1 H O P D A and 3,9,11-triCl H O P D A were fairly similar. B y contrast, the identity of the H O P D A influenced the reactivity of BphK with G S H . For example, the Km for G S H in the presence of 3,9,11-triCl H O P D A was ~3.5-fold higher than in the presence of 3-C1 H O P D A . In addition, the kC3X in the presence of 3-C1 H O P D A was 16X greater than in the presence of 3,9,11-triCl H O P D A . Primary plots of 1/v against 1/[C1 H O P D A ] revealed that the fitted lines intersected at a point on the negative side of the abscissa in the positive region of the ordinate, suggesting that binding of one substrate by BphK influenced binding of the second (Figure 26; Kiefer and Copley 2002). Table 16. Steady-state kinetic parameters of BphK for chlorinated H O P D A s Substrate Km CI HOPDA kcat (uM) (uM) (x 103 M V ) 3-C1 HOPDA 110(20) 17(5) 0.16(0.008) 9.5 (2.0) 3,9,11-triCl HOPDA 390(120) 10(3) 0.010(0.001) 1.0 (0.2) Values in parentheses represent standard errors. Experiments were performed using potassium phosphate, p H 6.5 (I = 0.05 M ) , 25 °C. -7Q0--200 --390-J [3-CI HOPDA] ((jM) •3se-i ^e-1 • 1 [3,9,11-triCI HOPDA] (MM) Figure 25. Steady-state analyses of the BphK-catalyzed transformation of 3-Cl H O P D A s . A . The rate of the reaction was determined using 50 u M (•), 100 u M (•) , 250 u M (A), 500 u M (•) and 1000 u M (O) G S H . The calculated parameters were: Km.Gsu = 110 ± 20 u M , Km.3Cu = 17 ± 5 and V = 0.88 ± 0.08 uM/min. B . The rate of the reaction was determined using 500 u M (•), 1000 u M (•), 1500 u M (•) , 2500 u M (O), and 3500 u M (A) G S H . The calculated parameters were: £ m . G S H = 390 ± 120 u M , J ^ - i r i C H = 10.1 ± 2.9 and V= 0.10 ± 0.008 ^M/min. Experiments were performed in potassium phosphate, p H 6.5 (I = 0.05 M ) , 25 °C and either 9.2 x 10" 8 M (A) or 1.7 x 10" 7 M (B) BphK. Data were analyzing using an equation describing a ternary complex mechanism and the least squares, dynamic weighting options of L E O N O R A (Cornish-Bowden 1995). The plotted lines were calculated at each [GSH] using the best fit parameters. -0.05 0.00 0.05 0.10 0.15 0.20 0.25 -0.005 0.000 0.005 0.010 0.015 0.020 0.025 1/[3-CI HOPDA] (|JM-1) 1/[GSH] (UM-1) Figure 26. Primary plots of steady-state the BphK-catalyzed transformation of 3-Cl and 3,9,11-triCl H O P D A s . The data are the same as those plotted in Figure 25. However, each plotted line was calculated by fitting the Michaelis-Menten equation to the data points collected at each fixed substrate concentration. A . 1/v vs l /[3-Cl H O P D A ] at 50 u M (•), 100 u M (•) , 500 u M (•), 1000 u M (0) and 2500 u M (•) G S H . B . 1/v vs 1/[GSH] at 10 u M (•) , 20 u M (•) , 35 u M (O), 50 u M (A) 3-Cl H O P D A . C. 1/v vs 1/[3,9,11-triCl H O P D A ] at 250 u M (•) , 500 u M ( A ) , 1000 u M (•), 1500 (O), 2500 u M (A) and 3500 (0) u M G S H . D. 1/v vs 1/[GSH] at 10 u M ( A ) , 20 u M (•), 35 u M (O), 40 u M (• ) and 50 u M (•) 3,9,11-triCl H O P D A . Experiments were performed in potassium phosphate, p H 6.5 (I = 0.05 M ) , 25 °C and either 9.2 x 10"8 M (A-B) or 1.7 x 10" 7 M (C-D) BphK Data were analyzed using the least squares, dynamic weighting options of L E O N O R A (Cornish-Bowden 1995). 4. DISCUSSION During the course of this research, the limitation of Bph enzymes for degrading PCBs was explored, an approach to overcome some of these limitations was established, and an apparent adaptation of the bph pathway to P C B degradation was investigated. More specifically, a comparison of four evolutionarily divergent extradiol dioxygenases resulted in the identification of recalcitrant P C B metabolites. To facilitate the engineering of enzymes to transform such metabolites, biological selections and high-throughput screens were developed. The high-throughput screen was used to increase the specificity of an extradiol dioxygenase for a model recalcitrant metabolite by -1000-fold. Finally, BphK, a GST of unknown function, was shown to catalyze the dehalogenation of an inhibitory P C B metabolite. These various contributions are discussed in more detail below. 4.1 Reactivity of evolutionarily divergent DHBDs The data presented in this PhD work expand on the published reactivities of DHBDLB400, D H B D P 6 - I and DHBDp 6 -III with mono-Cl D H B s and 2 ' ,6 ' -diCl D H B (Vaillancourt et al. 2003) and provide insight into the reactivity of T H B D R W I , whose physiological substrate, 2,2' ,3-THB, is structurally similar to 2 ' ,6 ' -diCl D H B , a recalcitrant P C B metabolite. The amino acid sequences of DHBDp6-I and THBDRWI are 53% and 36% identical to that of DHBDLB4OO, respectively. Similarly, the single-domain enzyme DHBD P 6 - I shares 17% sequence identity with the active site domain of DHBDLB4oo- The four studied enzymes possess markedly different reactivities towards PCB metabolites. Nevertheless, the data clearly indicate that each enzyme has greater substrate specificity for at least one chlorinated D H B than for unchlorinated D H B . As the data set is not exhaustive, it is likely that these enzymes cleave other polychlorinated D H B s with greater specificity as well. In most cases, chloro substituents had similar effects on the kinetic parameters of the different enzymes for D H B . Nevertheless, the effect of multiple chloro substituents on the reactivity could not be easily predicted from the reactivity of the corresponding mono-chlorinated DHBs . For example, the apparent specificity of DHBDp6-III for 4,3',5'-triCl D H B was over 12 times higher than for any of the corresponding mono- and di-chlorinated DHBs, and its Km was 25 to 50-fold lower. Overall, the current data agree reasonably well with steady-state kinetic parameters and relative activities previously reported by McKay et al. (2003). Both studies evaluated the steady-state kinetic parameters of D H B D P 6 - I and -III for D H B , 2'- and 3 ' -Cl D H B . The main differences are in the k°pp, kafp and partition coefficients of DHBDp6-I: in the current study they are up to an order of magnitude higher than previously reported. These effects are presumably due to the low iron content, as reported by the authors, of the enzyme used previously. McKay et al. (2003) also reported relative activities of 5 enzymes at 100 u M of various DHBs , by normalizing the data to the rate of cleavage of unchlorinated D H B . Accordingly, the relative activities at 100 u M D H B were calculated using the steady-state parameters reported in Table 6. For the 15 combinations of enzyme and chlorinated D H B examined in both studies, the relative activities agreed to within a factor of 3. Considering that McKay et al. (2003) isolated chlorinated DHBs from culture supernatants containing up to 20% unwanted chlorinated DHBs , this is a surprising degree of agreement. Nevertheless, the true measure of an enzyme's ability to utilize different substrates is the specificity constant (Fersht 1985). In this respect, the relative activities determined at 100 u M substrate do not agree well with relative specificities {kapp for each CI D H B normalized to kapp for D H B ) , differing by over two orders of magnitude in some cases. The correspondence between relative activity and specificity is particularly poor for substrates with low Km's, such as 3' ,5 '-diCl D H B . Related to this, despite investigating the relative activities of 5 enzymes for each of 17 DHBs , M c K a y et al. (2003) did not identify a single chlorinated D H B that is a better substrate for a given enzyme than D H B . The physiological roles of the single-domain D H B D s in R. globerulus P6, DHBDp6-II and DHBDp6-III, remain unclear. Based on the higher specificity of these enzymes for some chlorinated D H B s versus unchlorinated D H B , it has been suggested that they may have been recruited by R. globerulus P6 to improve its P C B degradation (McKay et al. 2003; Vaillancourt et al. 2003). However, the current results demonstrate that relatively high specificity constants for chlorinated DHBs are not unique to the single-domain D H B D s . Moreover, the activities of the latter enzymes are relatively low compared to the two-domain enzymes. Finally, conditions under which DHBDp6-III is expressed in R. globerulus P6 have yet to be identified (McKay et al. 2003). It is possible that the primary physiological role of DHBD P 6-II and -III is not biphenyl catabolism or P C B transformation. Rhodococcal strains in particular appear to contain large numbers of DHBD-type enzymes, with 7 or more reported in each of strains TA421 (Maeda et al. 1995), RHA1 (Sakai et al. 2002) and K37 (Taguchi et al. 2004). Although the genes encoding these enzymes have been annotated as bphC's, recent Northern hybridization analyses in K37 (Taguchi et al. 2004) and D N A microarray studies in R H A 1 indicate that most of them are not involved in biphenyl degradation (E.R. Goncalves, H . Hara, D.S. Aeschliman, M . Fukuda, L . D . Eltis, and W.W. Mohn, unpublished data). Indeed, one homologue in R H A 1 seems to be involved in sterol catabolism while two others seem to be involved in the catabolism of cholic acid (Wilbrink et al., unpublished). The relatively low Kapp values of the enzymes for polychlorinated DHBs compared to the mono-chlorinated DHBs or even the non chlorinated D H B suggest that the substrate-binding pockets of these enzymes can readily accommodate the chloro substituents. The structure of a DHBDLB40O:DHB binary complex revealed that the substrate-binding pocket of this enzyme is lined with hydrophobic residues (Han et al. 1995). With the exception of the catalytic residues, Phel87, Asn243 and Pro280 line the proximal part of the pocket around the hydroxylated ring of D H B , and V a i l 4 8 , M e t l 7 5 , Ala200, Phe202 and His209 line the distal part of the pocket around the non-hydroxylated ring (Figure 27 A ) . Structural modeling and sequence alignments (Eltis and Bol in 1996) suggest that the respective substrate-binding pockets of the other enzymes are lined with similar types of residues. For example, a model of the THBDRwi :2 ' , 6 ' -d iC l D H B binary complex indicates that Phel83, Asn243 and Pro282 line the proximal part of the pocket Figure 27. Stereo view of (A) the structure of the active site of DHBDLB400 bound to D H B (pdb file 1 K M Y ; Han et al. 1995) and (B) the modeled active site of T H B D R W I bound to 2 ' ,6 ' -d iCl D H B (obtained using Modeller 6v2). The residues lining the proximal and distal parts of the substrate pocket are represented in blue and pink, respectively. A ) The ligands of the ferrous iron (shown as a green sphere) are His 146, His210 and Glu260. The conserved catalytic residues are His 195, His241 and Tyr250. B) The ligands of the ferrous iron (shown as a green sphere) are His 147, His209 and Glu260. The conserved catalytic residues are H i s l 9 1 , His241 and Tyr250. Figure made using P y M O L v.0.98 (Delano 2005). and Va l l49 , Metl76, Serl96, Prol98, and His208 line the distal part of the pocket (Figure 27 B) . In the crystallographic and modeled structures, the ring carbons of the bound substrate are relatively unencumbered: no residue approaches to within 3.25 A of C4, C5 and C6 of the bound D H B in the DHBD L B40o :DHB complex. C 3 ' , C4 ' and C 5 ' of the distal ring are even less sterically hindered, being oriented towards the opening of the active site. Overall, the architecture of the substrate-binding pocket of D H B D ' s and the sequence data are consistent with the kinetic data. The failure of four evolutionarily divergent D H B D ' s to significantly cleave 2' ,6 '-diCl D H B (Vaillancourt et al. 2003) suggests that such an activity is rare in nature and may be inherent in the binding of this compound to the active site iron. In all D H B D : D H B structures determined to date, the distal ring is twisted with respect to the proximal ring. In the structures of the 2 ' -C l D H B and 2 ' ,6 ' -diCl D H B complexes of DHBDLB400, a chloro substituent is positioned such that it abuts the dioxygen-binding pocket and may hinder proposed attack of a iron-superoxo species at C2 (Dai et al. 2002). Nevertheless, it might be possible to develop 2' ,6 '-diCl DHB-cleavage activity using directed evolution. A critical parameter in the success of directed evolution is the enzyme(s) selected as parent(s). THBDRWI and DHBDp6-I might be good starting points due to their relatively low inactivation constant (j°pp) and high kacpp with 2 ' ,6 ' -diCl D H B , respectively. Alternatively, DHBD P 6-III might be a better candidate as it appears to be poorly evolved for DHBs. In particular, this enzyme possesses a relatively low, broad specificity for chlorinated D H B s and relatively high Km for 2 ' ,6 ' -diCl D H B . The recent structure of DHBDp6-III may provide insight into the molecular basis of this specificity (Bolin et al, unpublished). 4.2 Selection and Screen development. A number of biological selections and screens were developed to facilitate the application of directed evolution to the engineering of enzymes to transform otherwise dead-end, i f not inhibitory, P C B metabolites, some of which are described in the previous section. Biological selections and screens, developed in strains designated herein as " S H C " , were based on the detection of benzoate, an end product of the upper bph pathway. The reporter genes, encoding an antibiotic resistance or luminescence, were placed under the control of the Pm promoter, which is turned on by X y l S in the presence of benzoate effectors (Figure 13). The efficacy of these systems in E. coli appeared to be limited by the toxicity of D H B . D H B was not toxic to another host, P. putida. However, even in this strain, the system did not link the activities of THBDRWI and H O P D A hydrolase to the expression of the antibiotic resistance gene. Two biological screens, constructed using the luminescence reporter luxCDABE, also failed to detect PCB-degrading activities. Consequently, a simple and versatile imaging-based high-throughput colorimetric screen was developed. This strategy, which exploited the yellow colour of the extradiol ring-cleaved product, enabled the screening of over 105 clones per hour and discriminated variants with as little as two-fold difference in activity. Despite the ultimate failure of the biological selections, the developed systems possess a number of characteristics which suggest that they may function as intended were a less toxic, more stable metabolite targeted. Firstly, the selections were very stable and could be maintained in the host cell in the absence of a selective pressure, in contrast to plasmid-based systems (de Lorenzo 1994). This is because the genetic elements of the selection were integrated into the chromosome of the host cells using mini-transposons. Secondly, the size of the colonies was proportional to the concentration of benzoate effector in the medium, a feature which could potentially enable the rank ordering of enzyme variants. Thirdly, P. putida SHC7 showed no background growth after 66 hours incubation in the absence of the benzoate, indicating that the system was well regulated in that strain. This characteristic suggests that the selection of false positives would be minimized in that host. Finally, the engineered strains are readily adaptable to other regulatory systems and phenotypic markers. Potential reporters include the green fluorescent protein (GFP), its derivatives and various antibiotic resistance markers. Some of these alternative systems could also be coupled to a Fluorescent Activated Cell Sorting (FACS) analysis apparatus, thereby increasing the throughput of the screen. Similarly, the developed systems could be readily converted to negative selections using a lethal marker such as SacB, which is toxic to some hosts in the presence of sucrose (Steinmetz et al. 1983). The current results illustrate the complications that can arise due to the unpredictability of in vivo conditions. Notably, DFFB appeared to be toxic to E. coli. Spontaneous D H B -resistance clones of E. coli CC118Rif were grown in the presence of 400 u M D H B and used for the construction of additional systems but also failed to allow for the induction of antibiotic resistance on the selection medium in the presence of D H B (results not shown). H O P D A , the DHB-cleavage product, may also be toxic. Consistent with this possibility, liquid cultures of Pseudomonas and E. coli strains harbouring D H B D rapidly produced H O P D A upon addition of D H B , and excreted the product in the medium, as indicated by the yellow colour of the supernatant after harvesting the cells (results not shown). Other potential limitations were the levels of THBDRWI and H O P D A hydrolase activities in the cell. However, the latter were determined to be theoretically sufficient to produce intracellular benzoate concentrations in the millimolar range in less than 1 minute. It is possible that H O P D A was pumped out of the cell before the hydrolase could transform it to benzoate. Alternatively, the levels of THBDRWI and H O P D A hydrolase in the cells grown on the selection medium may be lower than those in cells grown in the presence of 100 u M benzoate. In particular, tetracycline and chloramphenicol are bacteriostatic antibiotics that inhibit protein synthesis (Walsh 2003) and may have inhibited the production of THBDRWI and H O P D A hydrolase in sufficient quantities. The instability of D H B appeared to also limit the success of the screens. Plates containing D H B developed a purple-brown coloration over time, indicative of oxidation and polymerization of the catechol (Graham 1978). Thus, a significant amount of D H B may have degraded before the cells could turn it over to benzoate. In L B liquid selection medium, the half-life of D H B was approximately 7 hours at 37 °C (results not shown). A selection medium prepared with 200 u M D H B would thus contain ~35 u M D H B after 18 hours of incubation. The latter concentration ought to elicit a response in E. coli SHC2 and SHC4, as 10 u M benzoate induced antibiotic resistance {vide supra). However, it is unclear whether D H B was stable enough to ensure adequate benzoate production in P. putida SHC7 as colonies took 42 hours to appear in the presence of 25 u M benzoate (Table 9). Indeed, the stability of D H B at the surface of the solid medium could be lower than in liquid medium due to higher oxygen concentrations. Limitations arising from the instability of D H B might be circumvented by producing D H B in vivo using biphenyl and the first two enzymes of the upper bph pathway. This was attempted using E. coli SHC2, E. coli SHC4 and P. putida SHC7, respectively, containing a plasmid carrying the upper bph pathway. However, biphenyl was toxic to E. coli and did not induce the antibiotic resistance in P. putida. Another physiological parameter that appeared to be critical to the proper functioning of the SHC systems was the intracellular levels of X y l S , which regulates the Pm promoter. For example, benzoate induced chloramphenicol resistance in E. coli S17.Rpir ( p C N B l C A T ) , but not in the corresponding chromosome-integrated system, E. coli SHC1. A major difference between the two systems was the number of copies of xylS in the cell. Thus, p C N B l C A T is expected to be present in 10-15 copies per cell as it contains a R 6 K origin of replication (Kolter et al. 1978). In contrast, E . coli SHC1 likely contained a single copy of the xylS construct. It has been reported that transcription from the Pm promoter is influenced by both the intracellular levels of X y l S and the presence of benzoate, the effector. Thus, in P. putida mt-2 containing the T O L pathway, X y l S is constitutively expressed at low levels (Gallegos et al. 1996). Under these conditions, Xy lS activates transcription from the Pm promoter upon binding benzoate. However, overproduction of X y l S leads to the stimulation of transcription from the Pm promoter in the absence of effector (Inouye et al. 1987; Mermod et al. 1987; Gallegos et al. 1996). These observations led to the hypothesis that X y l S exists in two conformations, one of which is active: the effector shifts the equilibrium toward the active conformation whereas the overproduction of X y l S would simply increase the concentration of the active conformation (Mermod et al. 1987). Hence, the levels of X y l S may have been sufficient for Pm regulation in E. coli S17.lA.pir ( p C N B l C A T ) but too low in E. coli S H C l . The constitutive resistance to chloramphenicol of E. coli S H C l containing pVLT43 and E. coli SHC2 incubated in the presence of 100 u M IPTG further suggests that the problem with E. coli S H C l was linked to the intracellular levels of X y l S . The persistent problems with the different selection systems prompted the development of screening systems using the luminescence reporter luxCDABE. While luminescence was readily induced by exogenously added benzoate in P. putida SHC11 and E. coli SHC 12, induction by benzoate produced in vivo from D H B was less apparent. In fact, D H B immediately quenched the signal of luminescencing colonies and also appeared to quench luminescence in liquid cultures. The mechanism of quenching is unclear as the lux system emits maximally at -490 nm (Meighen 1993) while D H B absorbs negligibly above 300 nm at neutral pH (Vaillancourt et al. 2002a). It is possible that D H B disrupts one or more of the enzymatic reactions leading to luminescence. Nevertheless, the luxCDABE cassette has been used in several other bacterial biosensors to detect a range of compounds including metals, nitrate, naphthalene, isopropyl benzene and even PCBs (Nivens et al. 2004). It might be possible to develop a biological screen using a different reporter. Notably, GFP has been used in a Pseudomonas-based biosensor to detect aromatic compounds such as benzene, toluene, ethylbenzene and phenol (Stiner and Halverson 2002). Interestingly, P. putida SHC11 and E. coli SHC12 responded very differently to the presence of benzoate. Thus, in P. putida SHC11, the luminescence peaked after three hours, while that in E. coli SHC12, although weaker, increased linearly until the end of the experiment, at 3 hours. This may be due to the Pm promoter, which can be activated by one of two sigma factors depending on the growth phase of the cells (Marques et al. 1999). Thus, the Pm recruits a , associated with heat shock, during the exponential growth phase and a during stationary phase (Marques et al. 1999). The behaviour of the XylS/Pm system was essentially identical in E. coli and Pseudomonas (Marques et al. 1999). Interestingly, the activity of the Xy\S/Pm system determined using P-galactosidase as a reporter was very similar to that observed in P. putida SHC11 (Marques et al. 1999). In the studies using the P-galactosidase reporter, the drop in transcription from the Pm promoter was linked to the switch to a s (Marques et al. 1999). It is thus possible that the switch in sigma factors was delayed in E. coli compared to Pseudomonas. Nevertheless, an alternative explanation for the drop in luminescence observed in P. putida is that the benABCD pathway, responsible for benzoate catabolism in that strain, may have been induced, causing the rapid depletion of the benzoate in the medium (Cowles et al. 2000). The latter scenario is however less probable as the benABCD pathway would likely be inhibited by catabolite repression on L B medium (Morales et al. 2004). Several genetically engineered selections have been reported in the literature. Such selections are typically based on complementation of auxotrophy or resistance to cytotoxic agents such as antibiotics. Using an auxotroph for P-branched amino acids as a screening host, the specificity of an aspartate aminotransferase for 2-oxovaline was increased 105-fold through several rounds of mutation and selection (Yano et al. 1998). Similarly, a direct selection for antibiotic resistance was used to increase the moxalactamase activity of a cephalosporinase by 540-fold in a single cycle of gene shuffling (Crameri et al. 1998). Nevertheless, biological selections have been limited by the ability to link the desired activity to a conditionally selectable phenotype. Improved understanding of cellular physiology, particularly the molecular basis of regulatory circuits, provides a burgeoning number of ways to link activities to selectable phenotypes through modification of regulatory components. Transcription regulators remain an attractive means to link the desired activities to cell viability due to their precise regulation of gene expression and their plasticity (Korner et al. 2003). Indeed, using directed evolution, transcription regulators could be designed to detect virtually any small molecule that was not toxic to the host. More recently, a number of additional genetic regulatory devices have been discovered. Bacterial mRNAs containing sequences binding small molecules metabolites, or "riboswitches", regulate gene expression at either the transcriptional or translational level (Tucker and Breaker 2005). A synthetic riboswitch was shown to activate protein translation upon binding of the target molecule theophyllin and could be adapted to generate a biological selection (Desai and Gallivan 2004). Such systems are simpler than gene regulation circuits and offer great promise for the design of selections as riboswitches can potentially be tailored to recognize an infinite array of small molecules (Desai and Gallivan 2004). The choice of host for the biological selection or screening systems has a great impact on the latter's potential usefulness for directed evolution. E. coli and Saccharomyces cerevisiae are the two most widely used host cells for directed evolution experiments, principally because they are among the best understood microorganisms and may be engineered using a plethora of molecular biology tools (Arnold and Georgiou 2003). A critical parameter for library creation is the transformation efficiency of the host cells by the D N A vector. This parameter dictated the choice of E. coli as original host for the biological selections presented in this work as libraries of >108 clones could be obtained by electroporation. As the genetics and physiology of other bacterial strains improves, the range of available hosts wi l l undoubtedly expand, enabling the use of additional regulatory networks and the engineering of more robust selection systems. Due to the persistent limitations of the developed biological selection and screening systems, efforts were redirected to develop a colorimetric screen for the directed evolution of extradiol dioxygenases. The imaging-based approach that was developed greatly simplified the visual inspection of colonies, enabling the screening of over 105 clones per hour. The latter represents a 10-fold increase in throughput compared to direct visual examination in this experimentor's experience. Importantly, the developed approach facilitated the initial rank-ordering of enzyme variants. Remarkably, this ranking correlated well with that based on the activities measured in microplates using the raw extracts (results not shown). With the availability of coloured filters, the described screening strategy can be easily adapted to any biocatalyst whose reaction can be coupled to a colorimetric change. Indeed, colorimetric changes at particular wavelengths (e.g. red and blue) would not require any image filtering, and the color intensity of active clones could be quantified directly. For example, the engineering of indigo- and indirubin-producing cytochrome P450 monooxygenases (Li et al. 2000) and the directed evolution of a P-fucosidase activity from the p-galactosidase (LacZ) of E. coli (Zhang et al. 1997) would have been facilitated by the present screening method. Finally, the described system could be extended to non-colorimetric reactions by coupling the desired enzymatic activity to the expression of a reporter such as LacZ, which produces a measurable indigo compound in the presence of 5-bromo-4-chloro-3-indolyl-|3-D-galactoside (X-gal). 4.3 Directed evolution of D o x G Using the imaging-based screen discussed in the previous section, a directed evolution experiment combining a single round each of error-prone PCR, saturation mutagenesis and DNA-shuffling, yielded a DoxG variant, DOXGSMA2, that cleaved 3,4-DHB with an apparent specificity constant that was 770-fold higher than that of the wild-type enzyme. Interestingly, the increase in apparent kcat was relatively modest (4-fold), despite the fact that the concentrations of 3,4-DHB used to screen colonies ought to have selected for this parameter. Extradiol dioxygenases catalyze the cleavage of preferred substrates with kcat and Rvalues of up to 1350 s"1 and 6.2 x 10 7 M " ' s " \ respectively (Vaillancourt et al. 1998; Fortin et al. 2005). These values suggest that the 3,4-DHB-cleaving activity of DoxG could be further improved by as much as two orders of magnitude. Nevertheless, engineering an increase of 770-fold in any enzyme's specificity constant is significant. Directed evolution studies typically report improvements of 10-50 fold (Valetti and Gilardi 2004), and there are few reports of improvements of two to three orders of magnitude (Cho et al. 2004). The success of the current study was due in part to the relatively effective, inexpensive screening method that was developed (vide supra). Selection of suitable clones was further facilitated by verifying ring-cleaving activities in raw extracts using a microplate reader and two different substrates: 3,4-DHB and D H B . Nevertheless, to prevent the elimination of potentially interesting variants, those demonstrating a >4 fold increase in activity were selected regardless of their relative ability to catalyze the cleavage of the two substrates. For example, variant IP33 showed an unchanged 3,4-DHB/DHB activity ratio yet demonstrated a 5-fold increase of activity towards 3,4-DHB. Significantly, the rates observed in the high throughput enzymatic assays performed on the raw extract correlated well with the kinetics performed using pure enzyme preparations. Considering the concentrations of substrate used in the assays, the relative activities determined using the microplate were within a factor of 2 of those calculated from the steady-state kinetic parameters obtained using purified enzyme preparations. Furthermore, the relative activities determined spectrophotometrically using the microplate assays and raw extracts prepared from 1 ml cell culture correlated well with the relative activities measured using a Clark-type electrode and raw extracts prepared from 100 ml cultures. For the DOXGSMA2 variant for example, the difference in relative activity towards 3,4-DHB was 11% and the difference between the 3 ,4-DHB/DHB activity ratio was 4% (not shown). Based on the crystallographic structure of the DoxG:3,4-DFIB complex, the residues that are substituted in the DOXGSMA2 variant are less than 9 A from the bound substrate (Fig. 18B). Two of the mutated residues (L190M and L242S) may interact with the distal ring of the substrate. Interestingly, the two sets of substitutions (L190M/S191W and L242S) influenced the enzyme's apparent specific activity towards 3,4-DHB in an anti-cooperative fashion, but influenced that towards 1,2-DHN and D H B in an antagonistic fashion. While Mildvan's analysis (Mildvan 2004) has not been applied to multiple substrates in a single enzyme, the substrate-dependent nature of the cooperativity between the residues is not surprising in light of the unique way in which each substrate interacts with the enzyme. Although the role of each substituted residue in substrate binding and catalytic turnover remains unclear, the nature of the substitutions (two bulkier residues on one side of the substrate and a smaller residue on the other, see Figure 18B) are consistent with a reshaped binding pocket that binds 3,4-DHB in a different conformation. Overall, the dramatic changes in specificity were produced by relatively conservative changes in the substrate pocket of the enzyme. The specificity of DOXGSMA2 differs from that of DoxG in two other interesting respects. First, DOXGSMA2 preferentially catalyzes the distal cleavage of 3,4-DHB (2.8-fold over DoxG). Hence, the presence of Ser at position 242 could stabilize a reaction intermediate arising during the distal cleavage of 3,4-DHB. Second, the specificity constant of DOXGSMA2 for 1,2-DHN is 7-fold higher than WT DoxG. By way of explanation, Met l90 of DOXGSMA2 may interact with the distal ring of 1,2-DHN to a lesser extent than that of 3,4-DHB. Although DoxG has been implicated in naphthalene catabolism, its specificity constant for 1,2-DHN is quite low, suggesting that this is not the enzyme's optimal substrate. Moreover, the relatively large substrate-binding pocket of DoxG (Neau 2004) suggests that the natural substrate of the enzyme could be a polyaromatic catechol. Indeed, although the dox pathway catabolizes naphthalene (Denome et al. 1993), this is not necessarily the pathway's optimal, or natural, substrate. The effect of the substitution of Arg for Gin at position 101 is noteworthy as this residue is located at the surface of the protein, more than 30 A away from the active site's iron atom. Despite this apparent remoteness, this residue had a significant effect on the enzyme's specificity (compare variants IP33 and B8 in Table 10). DoxG is a homo-octamer. Within this octamer, G l n l O l of one subunit is about 4 A away from Arg l79 of a neighbouring subunit (Fig. 18B). Arg l79 is located at the N-terminal end of an unstructured loop that may form a flap over the substrate-binding pocket. Arg l79 is also next to Tyrl78, the tyrosine residue that shifts upon substrate binding (Neau 2004). The effects of substitutions at residues apparently remote from the active site, such as G l n l O l in DoxG, are difficult to predict. Identification of the Q101R substitution exemplifies the power of more random engineering approaches such as directed evolution, as such substitutions are usually overlooked in rational design experiments. The successful engineering of DoxG may be due to the enzyme's relatively large substrate-binding pocket, a feature that could translate into increased substrate pocket plasticity, more permissive to changes in proximity of its active site. During the course of this doctoral research, a similar engineering strategy was applied to DHBDLB400 and THBDRWI with less success (unpublished results). Compared with DoxG, these enzymes have smaller substrate-binding pockets and specificity constants for D H B that are close to 2 orders of magnitude higher. Furthermore, the present experiment constitutes an additional argument supporting the idea that enzyme engineering could be facilitated by starting with enzymes characterized by low, broad specificities (Taylor et al. 2001). 4.4 Transformation of inhibitory PCB metabolites by BphK As part of the effort to characterize and recruit enzymes to expand the PCB-degrading capabilities of bacterial strains, BphK was purified and characterized. The bphK gene is found in some bph gene clusters and encodes a GST of unknown physiological function. The enzyme was expressed heterologously to approximately 60% of the total cellular protein in E. coli and purified to >99% homogeneity with an overall yield of >160 mg protein per liter of cell culture. Chloride ion and HPLC-based assays demonstrated that purified BphK catalyzed the dechlorination of 3-C1 H O P D A , 5-Cl H O P D A and 3,9,11-triCl H O P D A . The dehalogenation of 3-C1 H O P D A was coupled to the oxidation of two G S H equivalents. In contrast, H O P D A , 4-Cl H O P D A , and D H B s chlorinated on the hydroxylated ring were not transformed. The 4 - C B A dechlorination activity reported previously (Gilmartin et al. 2003) was confirmed in the present study, but was 2300-fold lower than that for 3-C1 H O P D A . These data, combined with previous results discussed below, strongly suggest that BphK was recruited by LB400 to transform P C B metabolites such as chlorinated H O P D A s (Figure 28). H O . X O O H HO^ X O O H 2 G S H __L BphK -1 x 104 M-1s-1 G S S G .o + H C I 3-CI H O P D A H O P D A Figure 28. Dechlorination of 3-CI H O P D A by BphK (see Figure 27B for additional details). PCBs have been present in significant quantities in the biosphere since the 1950s (Abramowicz 1990). The relatively low specificity constants, kA, of BphK for 3-CI H O P D A s are consistent with the notion that this enzyme was recruited relatively recently to the bph pathway to facilitate the catabolism of PCBs. However, 3-CI H O P D A is unlikely to occur in vivo because it would have to originate from 4-chlorobiphenyl. In particular, the first Bph enzyme, biphenyl dioxygenase, hydroxylates this congener on the non-chlorinated ring (McKay et al. 1997). Further metabolism yields 10-C1 H O P D A and 4 - C B A . The kA value of BphK for the physiologically more relevant 3,9,11-triCl H O P D A , predicted to be formed by the transformation of 4,3',5'-trichlorobiphenyl, was ~10 3 M ' V 1 . By comparison, tetrachlorohydro-quinone (THCQ) dehalogenase, a GST that is thought to have been recently recruited for the degradation of pentachlorophenol (PCP), has a kA of ~7 x 104 M ' V for T H C Q (Kiefer and Copley 2002). A more highly evolved GST, human maleylacetoacetate isomerase ( M A A I ) , possesses a specificity constant of 1.3 x 10 6 M ' V for maleylacetoacetate (Board et al. 2003). Several additional lines of evidence support the hypothesis that BphK has been recruited by some bacterial strains to facilitate the catabolism of PCBs. First, the compounds on which the enzyme acts, 3-CI HOPDAs , are poor substrates for BphD as demonstrated in purified enzymes from B. xenovorans LB400 and R. globerulus P6 (Seah et al. 2000; Seah et al. 2001) and whole cells (Furukawa et al. 1979). Second, deletion of bphK does not affect growth on biphenyl (Bartels et al. 1999), suggesting that it has no role in biphenyl degradation. Third, BphK is only found in some 6p/z-containing strains. Moreover, although BphK homologues were >95% identical at the amino acid sequence alignment level in 5 different bacterial strains, the nucleotide sequence was significantly divergent in the regions flanking the bphK genes (Bartels et al. 1999), suggesting independent recruitment events. Steady-state kinetic analyses performed using different concentrations of 3-Cl H O P D A s and G S H revealed that BphK utilizes a ternary complex mechanism, as has been reported for other GSTs (Armstrong 1991) including T C H Q dehalogenase (Kiefer and Copley 2002), which has been the subject of recent mechanistic studies (Kiefer and Copley 2002; Kiefer et al. 2002; Warner et al. 1996). However, the present data were not sufficient to distinguish between a rapid-equilibrium random-order ternary-complex and a compulsory-order ternary-complex mechanism, as the two are kinetically equivalent (Cornish-Bowden 1995). BphK is typical of other GSH-dependant reductive dehalogenases in that two equivalents of G S H are oxidized during the dechlorination reaction. For example, the dechlorination of 2,5-dichlorohydroquinone, catalyzed by L i n D from Sphingomonas paucimobilis UT26 during the degradation of y-hexachlorocyclohexane (lindane), involves the oxidation of two G S H equivalents to glutathione disulfide (GSSG) (Nagata et al. 1999). B y contrast, most members of the GST superfamily, including M A A I and maleylpyruvate isomerase (MPI), utilize one molecule of G S H during catalytic turnover (Armstrong 1991). T C H Q dehalogenase is a particularly interesting example as it catalyzes two different reactions: the reductive dechlorination of T H C Q and the isomerization of maleylacetone to fumarylacetone (Anandarajah et al. 2000). The former reaction requires two GSHs whereas the latter uses a single molecule. The reactions catalyzed by BphK and T C H Q dehalogenase, respectively, share a number of similarities that make it tempting to speculate on the mechanism of the former, both to provide insight into GST-catalyzed reactions in general and to guide future mechanistic studies of BphK. Recent work on T C H Q dehalogenase indicates that the mechanism involves a ketonization followed by the 1,4-elimination of HC1 from 2,3,5,6-tetrachloro-4-hydroxycyclohexa-2,4-dienone, forming trichlorobenzoquinone (TriCBQ). T r iCBQ is attacked by GS~, yielding 2,3,5-trichloro-6-S-glutathionyl-4-hydroxycyclo-hexa-2,4-dienone, which is then converted to trichlorohydroquinone (triCHQ; Figure 29A) and an enzyme-GSH conjugate. A second G S H molecule is consumed to regenerate the free enzyme via a thiol-disulfide exchange that also yields glutathione disulfide. In substantiating this mechanism, the authors specifically ruled out two other mechanisms (Kiefer and Copley 2002; Kiefer et al. 2002; Warner et al. 1996): (1) nucleophilic aromatic substitution (SnAr); (2) an SN2 mechanism involving the displacement of the chloride by the thiolate form of glutathione (GS~) (Fig. 29A). The SNAT mechanism is generally accepted for the dechlorination-coupled conjugation of glutathione to the reporter substrate C N D B by GSTs (Armstrong 1997) and had been originally proposed for T C H Q dehalogenase (McCarthy et al. 1996). O f the three mechanisms, 1,4-elimination is most typical of the mechanisms utilized by GSTs. For example, the reactions catalyzed by the GSH-dependent isomerases involved in the degradation of tyrosine ( M A A I ) and benzoate (MPI), respectively, appear to share common steps with those catalyzed by T C H Q dehalogenase (Kiefer and Copley 2002). H O P D A ' s are hydrolyzed by BphD in a reaction that shares intriguing features with the proposed mechanism of T C H Q dehalogenase. In particular, the reaction is initiated by a tautomerization of the enol to the keto form of the H O P D A (Henderson and Bugg 1997). The keto tautomer is very similar in structure to proposed intermediates in the T C H Q dehalogenation and M A A isomerization, and could be subject to an analogous nucleophilic attack by GS" at C-4. However, 3-C1 H O P D A can not be dehalogenated through a 1,4-elimination, as postulated for the T C H Q dehalogenase. It seems more likely that the BphK-catalyzed dehalogenation of 3-C1 H O P D A proceeds via either a simple addition of GS" followed by elimination of chloride or an SN2 mediated displacement of chloride at C-3 by GS" as depicted in Figure 29B. The subsequent release of H O P D A and regeneration of the free enzyme using a second equivalent of G S H would 1,4 elimination of HCI Figure 29. (A) The proposed mechanism for the T C H Q dehalogenase-catalyzed dechlorination of T C H Q . The upper and lower routes show the 1,4-elimination of HCI and SN2 displacement of the chloride by GS~, respectively. (B) Possible mechanisms for the BphK-catalyzed dehalogenation of 3-CI H O P D A . The upper mechanism proceeds through simple addition/elimination steps. The lower mechanism is similar to the SN2 reaction that was ruled out for the T C H Q dehalogenase reaction. proceed in a manner analogous to that proposed for T C H Q dehalogenase. Moreover, a similar mechanism could be outlined for the dechlorination of 5-C1 H O P D A , a reaction that was catalyzed by BphK with a specific activity very similar to that for 3-Cl H O P D A . Interestingly, the proposed mechanism also does not allow for the enzyme-catalyzed dechlorination in position 4, consistent with experimental observations. The apparently recent recruitment of BphK by the bph pathway to degrade PCBs is reminiscent of the PCP degradation pathway of Sphingobium chlorophenolicum. Several lines of evidence indicate that the latter has recently evolved through the patchwork assembly of two other catabolic pathways (Copley 2000; Tiirola et al. 2002). Notably, the flavin oxygenase of S. chlorophenolicum that catalyzes the initial hydroxylation of PCP possesses a remarkably broad and low specificity compared to other flavin oxygenases (McCarthy et al. 1997). Also, the reductive dehalogenase catalyzing the second and third steps of the pathway, T C H Q dehalogenase, may have originated from M A A I , normally involved in degradation of tyrosine. Indeed, T C H Q dehalogenase still catalyzes the isomerization of maleylacetone, an analoque of M A A (Copley 2000). Moreover, the dehalogenase is strongly inhibited by its substrates, T C H Q and tr iCHQ, further suggesting that the enzyme is not highly evolved (Copley 2000). Finally, the genes of the PCP pathway are dispersed throughout the genome of S. chlorophenolicum and their transcription is poorly regulated (Copley 2000). Assuming that the recruitment of bphK for P C B degradation is recent, its origin is unclear. Homologues sharing approximately 60% amino acid sequence identity have been reported in Cycloclasticus oligotrophus RB1 (Wang et al. 1996) and Sphingomonas yanokuyae B l (Bae et al. 2003). C. oligotrophus R B I , a marine bacterium, grows on a range of aromatic compounds, including biphenyl. However, the physiological role and genomic context of xylK, the bphK homologue, are unknown. In S. yanokuyae B l , the bphK homologue enabled faster growth on m-toluate (Bae et al. 2003). On-going genome sequencing analyses should continue to provide insights into the origin of bphK and the physiological role of its homologues. When present, bphK is located within the bph operon, upstream of the genes encoding the extradiol dioxygenase and H O P D A hydrolase. This indicates that the expression of BphK is linked to the expression of the other enzymes involved in the production or transformation of H O P D A metabolites (Vuilleumier and Pagni 2002). Nevertheless, the genetic association of BphK with a dioxygenase suggests that the former may also help protect the cell against ROS generated by oxygenases of the pathway, as observed in Ochrobactrum anthropi during degradation of 4-C1 phenol (Tamburro et al. 2004). Thus, the dehalogenation catalyzed by BphK could be a moonlighting activity that fortuitously provides an advantage to B. xenovorans LB400 during growth in PCB-contaminated soils. Unfortunately, the reported data are insufficient to evaluate whether BphK-containing strains degrade PCBs more completely or confer an improved ability to survive in the presence of these compounds. Disruption of bphK may help establish the role of the enzyme in P C B degradation. 4.5 Concluding remarks The work presented in this thesis describes the characterization of evolutionarily divergent extradiol dioxygenases which possess markedly different specificities for the studied CI DHBs . Nevertheless each enzyme has greater substrate specificity for at least one chlorinated D H B than for unchlorinated D H B . As the data set is not exhaustive, it is likely that these enzymes cleave other polychlorinated D H B s with greater specificity as well. Interestingly, none of the enzymes were able to catalyze the cleavage of 2 ' ,6 '-diCl D H B and 3,4-DHB, which suggests that engineering extradiol dioxygenases activities to degrade these metabolites could significantly improve the PCB-degrading capabilities of bacterial strains. Although the biological selections designed in this study failed to link the activity of PCB-degrading enzyme to the viability of the cell, the knowledge gained wil l undoubtedly be useful in designing improved selection systems. The developed high-throughput colorimetric screen during this PhD work was useful in evolving an extradiol dioxygenase, and could be adapted to engineer other biocatalysts. The directed evolution strategy used in the present work provides a good framework for the engineering of other PCB-degrading enzymes, including extradiol dioxygenases with improved activities against 3-CI catechol and 2 ' ,6 ' -diCl D H B , two inhibitory P C B metabolites (Dai et al. 2002; Vailancourt et al. 2002b). The characterization of BphK reported herein constitutes the first report of a dehalogenase activity of this enzyme against inhibitory P C B metabolites. Transient-state kinetic analyses, mutational studies and on-going, collaborative crystallographic studies should provide considerable insight into the mechanism of the dehalogenase reaction. A directed evolution strategy could also be used to improve the activity of BphK against 3-CI H O P D A s and other critical P C B metabolites that prove to be actual metabolic blocks. 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Degradation of polychlorinated biphenyl (PCB) metabolites : directed evolution and enzymatic fortuity
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