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Characterization of the molecular basis for polychlorinated biphenyls (PCBs) transformation by biphenyl… Gomez Gil, Leticia 2006

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CHARACTERIZATION OF THE MOLECULAR BASIS FOR POLYCHLORINATED BIPHENYLS (PCBs) TRANSFORMATION BY BIPHENYL DIOXYGENASE by Leticia Gomez G i l B . S c , Universidad Complutense, 2000 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Microbiology and Immunology) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A November 2005 © Leticia Gomez G i l , 2005 ABSTRACT Biphenyl dioxygenase (BPDO) is the first enzyme in the bph pathway, catalyzing the dihydroxylation of biphenyl and some polychlorinated biphenyls. The B P D O s of different bacterial strains possess different abilities to transform P C B s . To better understand the molecular basis of these different abilities, highly active preparations of four B P D O s were anaerobically purified and characterized: BPDOLB4OO from Burkholderia xenovorans LB400, BPDOB356 from Comamonas testosteroni B-356, and two engineered variants, BPDOng and BPDOmo. BPDO119 is a variant of BPDOLB400 containing four substituted residues: T335G, F336I, N338T, and I341T. These residues correspond to those found in BPDOB356, belong to region III identified by Mondello et al, and contribute to the substrate-binding pocket of the enzyme. BPDOmo contains these substitutions as well as A267S. Steady-state kinetics assays demonstrated that of the four variants, BPDOB356 had the highest apparent & c a t for biphenyl, which was 10-fold higher than that of BPDOLB400- B y contrast, BPDOLB4OO had the highest apparent substrate specificity for biphenyl and was 10-fold higher than that of BPDOB356- The steady-state parameters of BPDO119 and BPDOmo for biphenyl were intermediate between those of the two parental enzymes. The identity of the residue at position 267 had a greater effect on the parameters than the identity of the region III residues. In all variants, the consumption of oxygen was well-coupled to that of biphenyl. The abilities of the four variant B P D O s to transform P C B congeners were investigated using three different methods: (1) purified enzymes and individual congeners; (2) purified enzymes and a mixture of 8 congeners; and (3) whole cells and a mixture of 8 congeners. The results obtained by each method were consistent. Most strikingly, BPDOB356 transformed a greater number of congeners at a faster rate than the other enzymes. Previously unrecognized activities of BPDOB356 include the 2,3-dihydroxyation of 2,4,4'-triCl biphenyl as well as the 2,3- and 3,4-dihydroxyation of 2,6-diCl biphenyl. For B P D O L B400 and B P D 0 B 356 , the degree of uncoupling was inversely related to how well the congener was transformed. The P C B -transforming abilities of BPDO119 and BPDOmo were more similar to those of BPDOLB4OO- However, both showed improved ability to transform either para- (BPDO119) or weta-substituted congeners (BPDOmo). The crystal structures of B P D O L B 4 o o and BPDO119 further confirmed the role of residues in region III in the range of substrates accepted by B P D O . 11 TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS . iii LIST OF TABLES v LIST OF FIGURES vi ABBREVIATIONS vii ACKNOWLEDGEMENTS ix 1. INTRODUCTION 1 1.1. P C B contamination and remediation 1 1.2. Aerobic biotransformation of P C B s : the bph pathway 2 1.2.1 The bph operon 5 1.3 Dioxygenases 6 1.3.1 Ring-hydroxylating dioxygenases 6 1.3.2 Classification of ring-hydroxylating oxygenases 7 1.3.3 Proposed mechanism for ring-hydroxylating oxygenases 8 1.3.4 Coupling between substrate consumption and dihydroxylation 10 1.4 Biphenyl dioxygenase (BPDO) 11 1.4.1. Crystal structure of B P D O 12 1.4.2. Substrate preference of B P D O 15 1.4.3. B P D O engineering 16 1.5 A i m of this study 19 2. MATERIALS AND METHODS 20 2.1. Chemicals and reagents 20 2.2. Bacterial strains, plasmids and culture conditions 20 2.3. D N A manipulation 20 2.4. Purification of B P D O components 21 2.4.1 Terminal Oxygenase (bphAE) 21 2.4.2 Ferredoxin (bphF) 23 2.4.3 Reductase (bphG) 23 i i i 2.5. Analytical methods 24 2.6. Steady-state Kinetic measurements 25 2.7. H P L C and G C - M S analyses 25 2.8. Coupling measurements 26 2.9. Depletion of P C B mixtures by purified B P D O s 26 2.10. Whole-cell assays 27 2.11. Characterization of the 2,4,4' P C B degradation product 27 3. RESULTS 28 3.1. Purification and characterization of B P D O 28 3.2. Steady-state kinetic analysis and uncoupling constants for the dihydroxylation of biphenyl 30 3.3. The reactivity of B P D O variants with individual chlorinated biphenyls 33 3.4. Characterization o f the transformation product o f 2,4,4'-triCl biphenyl by BPDOB356 36 3.5. The depletion of a P C B mixture by purified B P D O s 37 3.6 The depletion o f a P C B mixture by whole cells 38 4. DISCUSSION 40 5. BIBLIOGRAPHY 54 i v LIST OF TABLES Table 1.1. Amino acid sequence identities of representative ring-hydroxylating oxygenases 12 Table 1.2. Comparison of BphA sequences at key positions in variants 119 and 1110.18 Table 2.1. Plasmids, strains and oligonucleotides used in this study 22 Table 2.2. Extinction coefficients of the purified BPDO components 24 Table 3.1. Purification of the oxygenases 29 Table 3.2. Kinetic parameters and coupling constants of BPDOB356> BPDOLB4OO, BPDO119 and BPDOmo using biphenyl as substrate 32 Table 3.3. The depletion of individual chlorinated biphenyls by purified BPDOs 33 Table 3.4. The reactivities of purified BPDOs with individual congeners 35 Table 3.5. The depletion of a PCB mixture by purified BPDOs 38 Table 3.6. The depletion of a PCB mixture by E. coli cells co-expressing £>P/JAELB4OO, bphAEuv or bphAEmo and Z?P/?FGBCLB4OO 39 v LIST OF FIGURES Figure 1.1. P C B structure and nomenclature 1 Figure 1.2. Pathways for the degradation of polyaromatic compounds 3 Figure 1.3. The biphenyl upper pathway 4 Figure 1.4. The bph operon in different bacterial strains 6 Figure 1.5. The proposed stepwise (A) and concerted (B) mechanisms for ring-hydroxylating dioxygenases 8 Figure 1.6. The proposed concerted mechanism for naphtalene dioxygenase 9 Figure 1.7. Proposed routes of uncoupling in ring-hydroxylating dioxygenases 10 Figure 1.8. Dihydroxylation of biphenyl by B P D O 11 Figure 1.9. The structural fold of B P D O B 3 56 13 Figure 1.10. The metallocentres of BphAE B356 15 Figure 3.1. S D S - P A G E of the purified components of B P D O 28 Figure 3.2. The U V - v i s absorption spectrum of oxidized bphAEng 30 Figure 3.3. Steady-state dihydroxylation of biphenyl by A ) BPDO356 and B) BPDO119 31 Figure 3.4 . The N M R spectra of the product of 2,4,4'-trichlorobiphenyl dihydroxylation by B P D O B356 37 Figure 4.1. The substrate-binding pocket of BphAELrwoo 47 Figure 4.2. Shifts in active site residues of BphAE L B4oo induced by biphenyl-binding. 48 Figure 4.3. A topology map o f BphA B356 depicting the relative positions of the determinants of substrate specificity 49 Figure 4.4. The substituted residues in BphAEng and BphAEmo 50 v i ABBREVIATIONS A T P Adenosine triphosphate B P D O Biphenyl dioxygenase BPDOLFMOO Biphenyl dioxygenase from Burkholderia xenovorans LB400 B P D O B356 Biphenyl dioxygenase from Comamonas testosteroni B356 B P D O R H A I Biphenyl dioxygenase from Rhodococcus sp. strain R H A 1 B P D OKF707 Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 BPDO119 Engineered biphenyl dioxygenase BPDOmo Engineered biphenyl dioxygenase B P H Biphenyl C D O Cumene dioxygenase C D O J S 3 7 5 Cumene dioxygenase Pseudomonas fluorescens IP01 D N T P Deoxynucleoside triphosphate D T T Dithiothreitol G C - M S Gas chromatography-mass spectrometry H P L C High performance liquid chromatography EPTG Isopropyl-beta-D-thiogalactopyranoside ISC Iron sulfur cluster assembly M E S 2-(N-morpholino)ethanesulfonic acid N A D H Nicotinamide adenine dinucleotide N B D O Nitrobenzene dioxygenase N B D OJS765 Nitrobenzene dioxygenase from Comamonas strain JS765 N D O Naphthalene dioxygenase N D O 9 8 1 6 4 Naphthalene dioxygenase from Pseudomonas strain NCTB 9816-4 N M R Nuclear magnetic resonance P A G E Polyacrylamide gel electrophoresis P C B Polychlorinated biphenyl v i i P C R Polymerase chain reaction U V Ultraviolet ACKNOWLEDGEMENTS I would like to thank my supervisor, Lindsay Eltis, for his trust and patience. Thanks for allowing me to do things "my way". Thanks to all past and present members of the Eltis lab for their help and frienship. I must especially thank Pascal Fortin and Geoff Horsman, for their valuable advice and comments on my research, and Sachi Okamoto and Thomas Heuser, for their constant support. Many thanks to my committee members, Tom Beatty and B i l l Mohn, for looking after me and for reviewing my thesis. I would also like to thank Diane Barriault and Miche l Sylvestre for allowing me to use their engineered enzymes and for their collaboration on some parts of this work. Many thanks to Pravindra Kumar and Jeffrey Bol in for providing such good quality crystal structures of various B P D O s and for their very interesting feedback on those. Thanks to Gord Stewart for his help on both G C - M S and G C - F I D . On a more personal level, I would like to thank all the friends I have made in Vancouver for making these three years a unique experience. Last, but certainly not least... M i l gracias a mi familia, en especial a mi madre, por su apoyo constante y su amor incondicional. Gracias tambien a los pocos pero queridisimos amigos que deje en Espana y que han sobrevivido a estos tres anos de distancia. Gracias a todos por estar ahi cuando se os necesita. Sin vosotros este master no hubiese sido posible. ix 1. INTRODUCTION 1.1. PCB contamination and remediation Polychlorinated biphenyls (PCBs) are man-made compounds (xenobiotics) produced by the direct chlorination of biphenyl. The production process results in up to 209 congeners, each differing in position and number of chlorine substitutions. Their nomenclature is based on the position of these substitutions on the phenyl ring (meta, ortho, para) or according to the numbering of the biphenyl carbons (Figure 1.1). P C B s have low water solubility, low electrical conductivity and are extremely heat and chemical resistant. These properties led to their wide spread use as flame retardants, plasticizers and insulating fluids. P C B s were originally synthesized and sold as complex mixtures of 60 to 90 congeners, such as Aroclor. The mixtures differ in the extent of chlorination and specific congener composition. Due to their nature, P C B s persist in the environment and accumulate in the food chain, causing adverse health effects in humans, such as liver and neuronal damage, alterations in the immune system and increased incidence of diabetes and cancer (36, 61, 77, 78). Although their production was banned worldwide in the 1980's, there was already widespread contamination of the environment. Meta Ortho Ortho Meta Para C l n H {10-n} 6' 5' Polychlorinated Biphenyl (PCB) Para Meta Ortho Ortho Meta Figure 1.1. PCB structure and nomenclature. Biphenyl molecule with the numbering and substitution system for the chloro substituents. In the environment, P C B s are generally concentrated on sediment and soil surfaces due to their low solubility in water (61). PCB-contaminated soils have been 1 treated by different methods (97) such as soil washing, solvent extraction or chemical dehalogenation (Base Catalyzed Decomposition Process, B C D P ) . The most common treatment is incineration, which can produce toxic by-products that need to be further treated. Emerging approaches include stabilization, vitrification and bioremediation. The latter is based on the use of microorganisms, which can be used on-site and which mineralize P C B s to non-toxic carbon dioxide, chloride and water. Microorganisms can transform P C B congeners anaerobically and aerobically. Anaerobic transformation is based upon microbial respiration (11, 58, 73, 76, 91). In respiration, microbes gain energy from the consumption (oxidation) of electron donors coupled to the utilization (reduction) of electron acceptors. Highly chlorinated P C B s act as electron acceptors because of their high oxidation state and chlorines are then replaced by hydrogen (63). This is a highly selective process and none of the microbial dechlorination patterns characterized, distinguished by congener selectivity and position of the removed chlorine substituents, show reductive dehalogenation of ort/zo-substituted chlorines. B y contrast, aerobic bacteria co-transform P C B s via catabolic pathways. Most of the isolated aerobic PCB-degrading bacteria can only transform up to tetra-chlorobiphenyls, although some of them can transform some congeners with up to six chlorine substituents (10, 66). However, commercial P C B mixtures usually contain congeners with more than six chlorine substituents. M u c h of the P C B content in contaminated sites is thus resistant to aerobic microbial mechanism. A potential way to overcome this is the sequential treatment of highly chlorinated congeners with both anaerobic and aerobic transformations. In a first step, anaerobic dehalogenation can potentially transform P C B s to less chlorinated congeners that are susceptible to a second step of aerobic microbial mineralization. 1.2. Aerobic biotransformation of PCBs: the bph pathway The pathways used by bacteria to aerobically degrade aromatic compounds are often arranged in "upper" and "lower" pathways encoded by genetically distinct loci (81). Upper pathways typically catalyze the activation, and sometimes the cleavage, of the aromatic substrates, transforming the latter to one o f a limited number of intermediates. Lower pathways convert these intermediates to T C A (Tricarboxylic A c i d Cycle or Krebs cycle) cycle precursors such as pyruvate or acetaldehyde. This organization of pathways enables the microorganism to maximize metabolic capacity while minimizing genetic redundancy. The activation catalyzed by the upper pathway 2 generally involves dihydroxylation, and typical intermediates include catechol, gentisate and protocatechuate (Fig. 1.2). clibenzo-p-dioxin difoenzofuran naphthalan© biphenyl gentisate pathway Figure 1.2. Pathways for the degradation of polyaromatic compounds. Reactions designated by an (a) are catalyzed by ring-hydroxylating dioxygenases. Reactions designated by a (b) are catalyzed by ring-cleaving dioxygenases. The intermediate obtained after the hydroxylation of benzoic acid depends on the substituent present on its aromatic ring. From (92). P C B s are aerobically co-transformed via the bph pathway, which is responsible for the catabolism of biphenyl (71, 81). The initial reaction in the upper pathway is catalyzed by biphenyl dioxygenase (BPDO), a multicomponent enzyme consisting of a 3 FAD-containing reductase (BphG), a Pvieske-type ferredoxin (BphF) and a two-subunit, hexameric oxygenase 0 ^ 3 (BphAE). B p h G and BphF transfer electrons from N A D H to B p h A E , which transforms biphenyl to cis-(2R,3S)-dihydrodihydroxy-l-phenylhexa-4,6-diene. The latter is dehydrogenated and then oxygenolytically cleaved by biphenyl-2,3-dihydrodiol dehydrogenase (BphB) and 2,3-dihydroxybiphenyl-l,2-dioxygenase (BphC), respectively. The ring cleavage product is then hydrolysed to benzoate and 2-hydroxy-2,4-dienoic acid by the hydrolase BphD (Figure 1.3). The lower bph pathway is comprised by a hydratase, an aldolase and a dehydrogenase, which successively transform the dienoic acid to acetaldehyde and pyruvate. Benzoate can be further catabolized to T C A cycle intermediates by up to six different pathways (Fig. 1.2) (26). Figure 1.3. The biphenyl upper pathway. B p h A E F G , biphenyl dioxygenase; BphB, biphenyl-2,3-dihydrodiol dehydrogenase; BphC, 2,3- dihydroxybiphenyl-1,2-dioxygenase; BphD, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase. Bacteria that aerobically transform P C B s include members from the genera Alcaligenes, Pseudomonas, Burkholderia, Comamonas, and Rhodococcus (13, 17, 37, 47, 59). Some of them can grow on mono- and dichlorinated biphenyls and most co-metabolize more highly chlorinated biphenyls using biphenyl as a growth substrate. The number and position of substituents in the biphenyl ring affect the rate of microbial transformation. Overall, lower chlorinated P C B s and P C B congeners with chlorines on one aromatic ring are preferentially transformed over higher chlorinated P C B s or P C B s with substituents on both aromatic rings. Moreover, less chlorinated congeners are usually transformed more completely. A s mentioned above, the degrading capacities of aerobic bacteria are very restricted. Engineering this pathway to degrade a greater variety of P C B s is one of the most promising approaches in bioremediation. Effective engineering requires better knowledge of the pathway and its regulation. 4 1.2.1 The bph operon At least five different configurations of the bph genes have been reported, which differ with respect to genetic organization and transcriptional regulation (Fig 1.4) (72). Of these, the three best characterized are exemplified by the clusters found in Burkholderia xenovorans LB400, Achromobacter georgiopolitanum (formerly Pseudomonas sp.) KKS102 and Rhodococcus sp. strain RHA1, respectively. In B. xenovorans sp. LB400 (64), the bph genes are organized in a chromosomal cluster that contains 6/>/zAEFGBCKHJLD. The bphK gene encodes a glutathione S-transferase (bphK) and bphHJI encode the enzymes involved in the lower pathway (71). This organization is also found in Pseudomonas pseudoalcaligenes KF707 (34). In both strains, the catabolic genes are preceded by a so-called orfO. The latter encodes a GntR-type transcriptional regulator that positively regulates its own expression and that of bphKHU in the presence of HOPDA and biphenyl (95). OrfO is also involved in the expression of the bphAEFG genes in LB400 (15). In KF707, a second regulator, BphR2, which belongs to the LysR family, activates the transcription of bphAEFGBC (94). The organization of the bph operon in A. georgiopolitanum KKS102 (51) also occurs in Comamonas testosteroni B356 (2), and is characterized by the location of the lower pathway genes upstream of the upper pathway genes. Moreover, the gene encoding the BPDO reductase, bphG, is localized at the end of the gene cluster, separated from bpKD by an orfl of unknown function. In KKS102, the regulation of the bph genes is dependent on bphS, the first gene of the cluster. BphS is a GntR-type transcriptional repressor and negatively regulates the pE promoter, which is located upstream of the bph operon. The binding of BphS is abolished in the presence of HOPDA (69). In contrast to the bph operons of LB400 and KKS102, the bph genes of Rhodococcus sp. strain RHA1 (59) are not found together at a single genomic location. This is also the case in other rhodococcal strains, such as M5 (70) and TA421 (53). In these strains, the genes encoding the first 3 enzymes of the pathway are found in a single cluster. In M5 and TA421, the gene order is bphAEFGBC. In RHA1, the order of bphB and bphC are reversed. The catabolic gene cluster is followed in all three rhodococcal strains by the bphS and bphT genes (termed bpdS and bpdT in strain M5), a two-component signal transduction system. BphS, the sensor kinase, is activated by biphenyl and other aromatic compounds, to phosphorylate BphT, the response regulator. 5 A deeper understanding of the bph operon and its regulation is crucial to engineer bacteria with improved P C B degradation capability. This w i l l facilitate development of a system to ensure strong induction under different environmental conditions. owa A > M aap 4.1 M a c K H 4 1 n 1 k b l ^ l l l J i l l l i l l > ^ P » r ^ ^ LB400 ORfP At A2 OR? A3 M B C D B N i m i i m i t s s s s r M ^ K F 7 1 5 lUUlUUD^B^m SS52HZ& I • ' D P RHA1 S £ C ^ 0«« /»» A3 A3 B C O Oftfj A4 < — ! F^^P? 1^^ —>HTrHm KKS102 t t lJ Oxygenase a-sutjunit Bij^ enyWhydrocliol dehydrogenase ES GlutalWsfi* transferase Oxygenase p-subunit E2^  2,3-Oihydr£>xybiphenyl 1,2-cioxygenase 2-Hydroxypen!a.2,<*.<iienoate hydrata&e B § Fftiradoxin d} HOPOA hydrolase O Acotakiehyde dehydrogenase fSB Oxittoreductnse (B) R«gulator g3 4-Hydrt!xy-2-e^alerate aMolasa Figure 1.4. The bph operon in different bacterial strains. The organization of the bph genes in the following bacteria: B. xenovorans LB400 , P. putida K F 7 1 5 , rhodococcal strains M 5 and R H A 1 and A georgiopolitanum K K S 1 0 2 . Taken from (72). 1.3 Dioxygenases Dioxygenases are enzymes that catalyze reactions in which both atoms of dioxygen are incorporated into the product (18). Three general classes o f dioxygenases have been described: lipoxygenases catalyze the hydroperoxidation of polyenes; ring-cleaving dioxygenases catalyze the carbon-carbon bond fission of catecholic compounds; and ring-hydroxylating dioxygenases catalyze the cz's-dihydroxylation of arenes. 1.3.1 Ring-hydroxylating dioxygenases Ring-hydroxylating dioxygenases, of which B P D O is a typical example, contain an iron-sulfur cluster and a non-heme iron. Both metal centres are located in the a subunit of the oxygenase. Iron-sulfur clusters are common in proteins involved in biological oxidoreductive functions (14). Although they can have sensory, structural, and even catalytic functions, their most common function is electron transfer (57). The chemical versatility of sulfur and iron are ideal for accepting, donating, and storing electrons. 6 Iron-sulfur clusters occur in one o f four configurations in biology: as a mononuclear Fe ligated to 4 cysteines, rhombic [2Fe-2S], cuboidal [3Fe-4S], and cubane [4Fe-4S] clusters (14). The ligands of the [2Fe-2S] cluster are most commonly four cysteines. However, in the Rieske-type [2Fe-2S] clusters the ligands are two histidine (His) residues and two cysteine (Cys) residues. Formally, each iron center in a cluster can be ferric or ferrous. Therefore, [2Fe-2S] appear to have three possible oxidation states. Under physiological conditions, only two redox states are observed, with overall charges of 2+ and 1+, respectively (57). In Rieske-type clusters, the redox active iron is coordinated to two His residues. Two groups of Rieske FeS proteins have been recognized: those possessing high and low reduction potentials, respectively. High-potential Rieske proteins, typified by the cytochrome be \ complex of the respiratory chain, have pH-dependent reduction potentials, attributed to coupled deprotonation of the His ligands. On the other hand, low-potential Rieske proteins, such as BphF, have p H -independent potentials (24). The non-heme iron centres are also involved in many different biological reactions. They employ a variety of electron sources, such as metal-bound substrates or cofactors, to reduce oxygen to the peroxide oxidation state (41). This requires a flexible metal-coordination environment that allows binding of exogenous ligands and is usually achieved by the 2-his-l-carboxylate facial triad. This triad anchors the iron to the active site and maintains three additional czs-oriented sites, used to bind substrate and/or O2 (41). 1.3.2 Classification of ring-hydroxylating dioxygenases Ring-hydroxylating dioxygenases are similar in their structure, mechanism and cofactor requirements. They were first classified in three classes, based on the number of components and the nature of their redox centers (9). However, some oxygenases are difficult to classify according to this system. A more logical classification system is based on the sequences of the cc-subunit of the oxygenase, which reflects its phylogeny (68, 96). Both systems allow the classification of any dioxygenase within groups possessing certain catalytic characteristics and specificity. Moreover, both classification systems have identified four major subfamilies of ring-hydroxylating dioxygenases that preferentially transform toluene/biphenyl, naphthalene, benzoate and phthalate, respectively. 7 1.3.3 Proposed catalytic mechanism of ring-hydroxylating dioxygenases In the proposed catalytic mechanism of ring-hydroxylating dioxygenases (reviewed in (21), Figure 1.5), the mononuclear Fe center and the Rieske cluster of the enzyme start in the oxidized state. The activation of dioxygen by the dioxygenase requires binding of the aromatic substrate close to the mononuclear Fe center and the reduction of both metallocenters. The binding o f O2 to the reduced mononuclear iron generates a superoxide intermediate. This intermediate has been proposed to react in one of two ways. In the stepwise mechanism (A), the superoxide reacts with the aryl substrate to yield a bridged iron-alkyl peroxo species. Fission of the 0 - 0 bond yields a mono-hydroxylated alkyl species and an iron (V) species which would effect the second dihydroxylation on the same face of the alkyl substrate. In the concerted mechanism (B), the cleavage o f the 0 - 0 bond cleavage occurs first, yielding an 0=Fe(V)-OH intermediate that would effect dihydroxylation in a single step. Figure 1.5. The proposed stepwise (A) and concerted (B) mechanisms for ring-hydroxylating dioxygenases (from (21)). 8 More recent studies have yielded the crystal structures of ternary complexes of naphthalene dioxygenase (NDO) from Pseudomonas sp. N C I B 9816-4 with substrate and dioxygen (49). The dioxygen binds side-on to the iron (II) center, with iron-oxygen distances of 1.8 and 2.0 A. The aryl substrate is positioned slightly further from the iron, with distances of 3.3 A (C2) and 2.9 A (C3) from the bound oxygen. The structures of N D O with its substrates support a concerted mode of attack, in which both oxygen atoms are polarized similarly and react with the carbon atoms of the substrate double bond (Fig. 1.6). This reaction would explain the cz's-stereospecific addition of both oxygen atoms to substrates by N D O and other dioxygenases. y V V „ Ferredoxin N o — T * C u e -(1) Ferredoxin //Product e-[2Fe2S]( OH.H (6) 2H + Air oxidation [2Fe2S]; N O — F s -(3) Substrate •o i r v [ 2 F e 2 S ] ^ (5) (2) Substrate (4) Figure 1.6. The proposed concerted mechanism for naphthalene dioxygenase. 1, the resting enzyme with oxidized Rieske center and ferrous active site; 2, the reduced enzyme; 3, binary dioxgen complex; 4, binary substrate complex; 5, ternary substrate dioxygen species; and 6, product naphthalene czs-l,2-dihydrodiol. Taken from . 9 1.3.4 Coupling between substrate consumption and dihydroxylation Reactions catalyzed by oxygenases are tightly coupled, meaning that every reducing equivalent, e.g. N A D H in B P D O , is used to transform the aromatic substrate. Thus, the transformation of the latter is coupled to the consumption of 0 2 . Uncoupling occurs when the activated oxygen is unable to react with the aromatic substrate because of steric and/or electronic impediments within the active site. The reducing equivalents are then used to produce peroxide radicals from the activated oxygen (Figure 1.7). Uncoupling is usually caused by the binding of a poor substrate or a substrate analogue that induces the same changes in the active site as a good substrate, but is unable to react with the activated oxygen. The process has been relatively well characterized in cytochrome P450 c am, in which the transformation of camphene, an analogue of camphor, is uncoupled from the oxygen consumption. Crystallographic studies indicate that camphene is highly mobile in the active site, allowing the entry of solvent. The latter acts as a source of protons, leading to the production of H2O2 and/or H2O from the activated Figure 1.7. Proposed routes of uncoupling in ring-hydroxylating dioxygenases. The nature and number of activated oxygen intermediate(s) are unknown. The formation of O2", H2O2 and H2O consume 1, 2 and 4 reducing equivalents, respectively. 0 2 (74). 10 The investigation of the relative degree o f uncoupling with different substrates provides a valuable insight in the binding of the different P C B substrates to the active site. Uncoupling in B P D O B356 and B P D O L B4OO by certain P C B congeners proceeds mostly via formation of H2O2 (48, 60). This production o f H2O2 has also been observed in N D O (56) using benzene as a substrate, suggesting a similar mode of action in both enzymes. 1.4 Biphenyl dioxygenase (BPDO) B P D O is the first enzyme in the biphenyl degradation pathway and is a typical three-component, ring-hydroxylating dioxygenase. A s summarized in Figure 1.8, the enzyme catalyzes the insertion of molecular oxygen into the aromatic substrate nucleus forming cw-(2i?,35 f)-dihydroxy-l-phenylcyclohexa-4,6-diene. Each a subunit o f 013P3 oxygenase contains a Rieske-type Fe2S2 cluster and a mononuclear iron center, which represents a total of 3 [2Fe-2S] and 3 mononuclear iron centers per holoenzyme. Electrons flow sequentially from N A D ( P ) H to the F A D center in BphG, the Rieske iron sulfur cluster in BphF and the Rieske center in the terminal oxygenase (50). The mechanism of dihydroxylation is thought to very similar to that of N D O from Pseudomonas sp. N C I B 9816-4 (see section 1.3.3), the best characterized ring-hydroxylating dioxygenase (40). 0laxyg«wM NADH + H* Figure 1.8. Dihydroxylation of biphenyl by BPDO 11 1.4.1. Crystal structure of BPDO The first published crystal structure of a ring-hydroxylating oxygenase was that of N D O from Pseudomonas strain N C I B 9816-4 (50). To date, the published crystal structures of related oxygenases include those of cumene dioxygenase from Pseudomonas fluorescens IP01 (CDO), nitrobenzene dioxygenase from Comamonas strain JS765 ( N B D O ) and B P D O from Rhodococcus strain sp. R H A 1 (27, 30, 35). The crystal structures of the B P D O oxygenases from C. testosteroni B356 ( B p h A E B 3 5 6 ) and B. xenovorans LB400 (BphAELB4oo) have been solved, but not published (1, 48). B P D O s from B. xenovorans LB400 and P. alcaligenes KF707 have also been modeled based on the N D O structure (87, 99). The available crystal structures reveal that the different oxygenases have very similar structural folds and subunit conformation, consistent with the high identity of their respective amino acid sequences (Table 1.1). The fact that the enzymes share very similar structures further supports the hypothesis that they also share a similar mechanism. Enzyme B P D O B 3 5 6 B P D O L B 4 0 0 B P D O K F 7 0 7 B P D O R H A I C D O J S 3 7 5 N D 0 9 8 1 6 4 NBD0 J S 76S BPDOLB4OO 75 100 95 68 74 33 32 B P D O B 3 5 6 100 76 76 66 76 33 33 Table 1.1. Amino acid sequence identities of representative ring-hydroxylating oxygenases. Values represent the percent identities o f amino acid sequences over the entire length of the large and small subunits. Each sequence is identified according to the strain and dioxygenase to which the oxygenase belongs. The quaternary structure of the 013P3 oxygenase resembles a mushroom (Fig. 1.9) in which the a subunits, arranged in a trigonal symmetry, form the cap and the P subunits form the stalk. Within a given a subunit, the Rieske-type [2Fe-2S] cluster and mononuclear iron center are separated by 43 A. However, the [2Fe-2S] cluster from one subunit interacts with the mononuclear iron center o f the neighbouring subunit, at a distance of 14 A. 12 Figure 1.9. The structural fold of BPDOB3 5 6 - The quaternary structure resembles a mushroom in which the three a subunits (blue) form the cap and the (3 subunits (brown) form the stalk. Purple and orange spheres represent the iron and sulfur atoms, respectively. The ligands of the metallocentres are completely conserved among oxygenases. In BphAE B356, the ligands of the Rieske-type cluster are CyslOO, C y s l 2 0 , H i s l 0 2 and His 123, all o f which are located in the N-terminal half o f the a subunit. The sulfur atoms in the Rieske-type cluster are hydrogen bonded to A r g l 0 3 , M e t l 0 5 , H i s l 2 3 and Trp l25 , conserved among various B P D O s . In N D O , however, the residue corresponding to M e t l 0 5 is Lys . The ligands of the mononuclear iron in BphAEB356 are His233, His239 and Asp388, all o f which are located in the C-terminal half of the a subunit. The Asp can apparently ligate the iron in different ways: in BphAERHAi it is monodentate (35) whereas in N a h A E (50) and BphAE B356 (1) it is bidentate. In N a h A E , a fourth residue, Asn201, 13 occurs close to the mononuclear iron. The distance of 3.8 A is too great for A s n to be a ligand. However, this residue could protonate the proposed peroxo intermediate (49). Mutation of the equivalent residue in BphAEe356, Gln226, by A l a , G l u and A s n indicated that it is not required for coordination of the iron, since all mutants were active (1). However, the substitution Gln226Glu led to a high degree of uncoupling and substrate inhibition when biphenyl was used as a substrate. This suggests a role of Gln226 in product formation/stabilization. Particular structural similarities between the B P D O s and N D O strongly suggest that these enzymes share highly similar catalytic mechanisms. First, the relative positions of the metallocentres in the oxygenases are very similar and are spanned by a conserved Asp (230 in BphAEB356)- This suggests that the electron transfer between the [2Fe-2S] cluster and the mononuclear iron occurs in the same manner. Second, the space between the Fe ion and the bound substrate in N D O corresponds to the dioxygen-binding site in N D O (49). In BphAERHAi, a dioxygen molecule can be accommodated in the corresponding space (35). Nevertheless, a series of conformational changes occur upon substrate binding in BphAERHAi that were not observed in N D O . Interestingly, most of these changes occur around just one of the biphenyl rings, making the substrate binding pocket larger and more accessible. Further conformational changes would also be required to bind P C B s , since they would otherwise not fit in this binding pocket. It is possible that the binding of naphthalene does not induce such conformational changes in N D O because the former is small enough to fit in the enzyme's binding pocket. 14 Figure 1.10. The metallocentres of BphAEB356- The mononuclear iron centre is coordinated by Asp388, His233 and His239, whereas one iron o f the Rieske cluster is coordinated by C y s l 0 0 and C y s l 2 0 and the other is coordinated by His 102 and His 123. Purple and orange spheres represent the iron and sulfur atoms, respectively. Further details about other residues are described in the text. 1.4.2. Substrate preference of BPDO A s the first enzyme in the bph pathway, B P D O is a major determinant of the spectrum o f P C B congeners that are transformed by a given organism. Studies on B P D O s from different organisms have revealed major differences in (a) congener (substrate) preference and (b) the mode o f ring hydroxylation. Bacterial strains were first classified based on their substrate preference by Bedard et al. (10) in 1990. The authors attributed differences in the degradation rates of various P C B congeners to the substrate preferences o f B P D O . Classes I and II preferentially transformed meta- over para- substituted congeners. In class II B P D O s , the chlorination of the non-hydroxylated ring did not influence congener reactivity. Class III preferentially transformed para- over ortho-15 substituted congeners; and class IV enzymes, represented by those of B. xenovorans LB400 and Ralstonia eutropha H850, degrade a broad range of congeners and exhibit both 2,3- and 3,4-dihydroxylation activity. The reactivity preference of class IV enzymes was ortho > meta > para chlorobiphenyls. It is now common to classify B P D O s as having either KF707- or LB400-type reactivity. These two B P D O s have been extensively studied and show very different reactivities (37). B P D OLB400 transforms a wide range of congeners, including some containing up to six chlorines (17, 65). A s noted above, B P D OLB400 preferentially transforms ort/zo-substituted congeners and catalyzes the 3,4-dihydroxylation of certain congeners with chlorines at positions 2 and 5, such as 2,2',5,5'-tetraCl biphenyl. BPDOLB4OO is also remarkable in that it catalyzes the dehalogenation of certain 2-C1 congeners (38, 80, 82, 83). The product o f this reaction is a 2,3-dihydroxybiphenyl (DHB) , obviating the need for BphB. 2,2'-Difluoro-, 2,2'-dibromo-, 2,2'-dinitro- and 2,2'-dihydroxybiphenyls are also substrates for the enzyme (80). B y contrast, B P D OKF707 transforms a narrow range of substrates and catalyzes neither 3,4-dihydroxylation nor ortho-dechlorination. Moreover, it preferentially transforms 4 ,4 ' -d iCl biphenyl over either 3,3'- or 2 ,2 ' -d iCl biphenyl (37). In addition to the fundamentally different reactivities of BPDOLB4OO and BPDOKF707, the regiospecificity and efficiency of transformation depends in a complex fashion on the substitution patterns of both aryl rings (83). Indeed, it has been suggested that the enzyme's substrate preference depends more on the relative position of the chlorines than on their total number (3). 1.4.3 B P D O engineering Protein engineering approaches, including structure-guided design and directed evolution, help identify the determinants of substrate preference in B P D O s and have generated variant enzymes possessing enhanced PCB-transforming activities. Such studies have been facilitated by the very similar amino acid sequences of BPDOLB4OO and B P D OKF707 despite their different reactivities. Thus, their respective BphFs and BphGs have identical sequences whereas B p h A and BphE share 95.6% (20 differences in 460 residues) and 99.5%o sequence identity, respectively (29, 88). Mondello and Erickson compared the sequences of BphA LB400 and BphAicF707 and used directed mutagenesis to identify four regions (I, II, III, and IV) whose sequences influence the range of congeners attacked (66). A l l four regions occur in the carboxy terminal half o f B p h A . Region III comprises seven residues that include Thr335, Phe336, Asn338 and Ile341. Substitution of individual residues in region III of BphALB400 improved the ability of the enzyme to 16 transform 4,4 ' -d iCl biphenyls, although the highest improvements in activity were achieved by multiple substitutions in this region, suggesting a cooperative or additive effect. The importance of these residues has since been confirmed by Kimura et al. and Barriault et al. (4, 6, 52). Also , resolution of the crystal structure of B p h A E revealed that residues in region III are located in the upper part of the substrate pocket. Kimura et al. also reported an expansion of the degradation capability of KF707 when Thr376 (region IV) was replaced by the corresponding residue in L B 4 0 0 , A s n (52). Although the protein engineering studies largely confirm that major determinants of substrate recognition are in BphA, there are also reports that BphE, the small subunit of the oxygenase, can influence substrate preference to some extent (33, 42, 52). Suenaga et al. developed a three-dimensional model of B P D OKF707 based on the crystal structure of N D O and created a series of site-directed mutants with changes in amino acid residues located close to the catalytic non-heme iron center, including residues 335 and 341 (87). Replacement of Ile335 by the corresponding Phe from L B 4 0 0 allowed the 3,4-dihydroxylation of 2,2',5,5'-tetraCl biphenyl by the mutant enzyme. Interestingly, the Phe227Val and Phe377Ala mutants exhibited a novel transformation of 3,3 ' -diCl biphenyl: 2,3-dihydroxylation appeared to be followed by a dechlorination yielding 3 ' - C l D H B . Similar studies have been carried out with models of B P D OLB400, proving that residues that are not in the immediate vicinity of the substrate can significantly influence the structure o f the active site (98, 99). Directed evolution, which mimics the natural evolutionary processes of genetic mutation, recombination and selection (25), has been applied to great effect in B P D O . Shuffling o f bphAiBm and bphA^joj yielded variants of the enzyme with very different activities and also supported the important role of regions III and IV in determining substrate preference (52). Gene shuffling between the L B 4 0 0 and the B356 oxygenases has also confirmed the important role of the C-terminal portion of the a subunit (5). Two of the variant enzymes characterized in this thesis, BphAEng and BphAEmo, were previously created by shuffling a targeted region of bphAmse and 6/?/JALB4OO (4). BphAEn9 and BphAEmo essentially have the same sequence as BphAELB4oo except that region III is replaced with that of BphAB356- The two variants differ by a single residue at position 267 (Ala in BphAE LB400 and BphAEng, and Ser in BphAE B356 and BphAEmo; Table 1.2). BphAEn9 showed an improved degrading capacity against all the congeners tested (4). B y contrast, BphAEmo was not able to transform any of them. More recently, Barriault et al. constructed a library of B P D OLB400 mutants in which some of the variants 17 exhibited a different regiospecificity against 2 ,2 ' -d iCl biphenyl, with 3,4-dihydroxylation preferred over 2,3-dihydroxylation and subsequent dechlorination (6). This study proved the influence of the residues in region III not only in the overall activity of the enzyme but also on its regiospecificity toward different congeners. Residue numbering 267 335 336 338 341 BphALB4oo A l a Thr Phe A s n He BphAn 9 A l a G l y He Thr Thr BphAmo Ser G l y He Thr Thr BphAB356 Ser G l y He Thr Thr Table 1.2. Comparison of BphA sequences at key positions in variants 119 and 1110. The table shows only those residues that were substituted in BphAng and BphAmo with respect to BphA LB400 (4). Thus, the sequence of BphAmo and BphAng at these positions is identical to that of BphAB356-The fact that relatively small changes in these highly conserved sequences of the LB400 and KF707 enzymes result in significant changes in substrate preference makes B P D O one .of the best targets for engineering bacteria with better PCB-degrading capacities. Engineering of B P D O s has not only generated enzymes that are better able to degrade P C B s , but also variants able to transform non-natural substrates, such as toluene, benzene and indole (54). A mutated version of B P D OKF707 catalyzed the angular dioxygenation of dibenzofuran, an uncommon dioxygenase activity (86). Changes in the enzyme's regiospecificity are also of great interest for organic synthesis. Thus, engineering of biphenyl dioxygenases shows a great potential not only for bioremediation, but also for so-called "green chemistry". 18 1.5 Aim of this study Given its importance in the bph pathway, B P D O is an attractive system to engineer. The overall objective of this study was to characterize four variants of this enzyme to gain a better understanding of B P D O and to facilitate its engineering for improved P C B degradation. The variants include two wi ld type enzymes, BphAE L B4oo and BphAE B356, and two variants generated via directed evolution, BphAEng and BphAEino (4). The steady-state kinetic constants of the purified non his-tagged enzymes for biphenyl were determined, and their activities towards various P C B congeners were investigated. Whole-cell assays were performed to compare the activity o f the enzymes in vivo to their activity in vitro. The degree of uncoupling between O2 utilization and congener transformation was determined for the different enzymes using different congeners. P C B transformation products for some of the most persistent congeners tested were characterized using mass spectrometry and N M R . Variant 119, B356 and LB400 were also used for crystallization studies, in the absence and presence of P C B congeners, to investigate the structural basis for changes in enzyme function. This is the first study in which the activities of anaerobically purified B P D O s , both engineered and wi ld type, are tested against against a range of individual congeners as wel l as against a mixture of congeners. The results are discussed in terms of the proposed catalytic mechanism of ring-hydroxylating dioxygenases and the crystal structure of B P D O . 19 2. MATERIALS AND METHODS 2.1. Chemicals and reagents Biphenyl was purchased from Aldr ich (Mississauga, ON) . P C B congeners were either purchased from Accustandard (New Haven, U S A ) or were kindly provided by Dr. Victor Snieckus (Queens University, Kingston, ON) . Restriction enzymes and T4 D N A polymerase were purchased from New England Biolabs (Pickering, ON) . Pwo D N A polymerase was purchased from Roche (Laval, QC) . Oligonucleotides were purchased from the N A P S Service unit at the University of British Columbia (Vancouver, B C ) and IDT (Integrated D N A Technologies, Coralville, U S A ) . Acetonitrile, ethyl acetate and hexane (Fisher Scientific, Mississauga, ON) were of H P L C grade. A l l other chemicals were o f reagent grade or better. 2.2. Bacterial strains, plasmids and culture conditions The strains and plasmids used in this study are summarized in Table 2.1. Plasmids pDB31-II9 and pDB31-II10 for cloning of bphAEng and bphAEmo, respectively, were generously provided by Dr. Miche l Sylvestre ( INRS-IAF, Montreal, QC) . Strains harbouring pT7 derivatives were grown in the presence of ampicillin (100 ng/mi) or carbenicillin (15 ag/ml). Strains harbouring pPAISC-1 were grown in the presence o f tetracycline (20 ng/ml). For B P D O expression, E. coli strain C41(DE3) (62) harbouring the isc plasmid (1) and the appropriate pT7 vector was grown in Terrific Broth (90) supplemented with 0.1 mg/ml ferric ammonium citrate. Culture media were inoculated with 1% (v/v) o f an overnight culture and grown at 37°C until OD600 reached 0.9-1.0. Expression of the bphAE genes was then induced by the addition of isopropyl-l-thio-P-D-galactopyranoside (IPTG) to a final concentration of 1 m M and the culture was transferred to 20°C (LB400, 119 and 1110) or 25°C (B356) for an additional 18 hours before harvest by centrifugation. Ce l l pellets were frozen at - 8 0 ° C until purification. 2.3. DNA manipulation D N A was digested, ligated and transformed into E. coli using standard protocols (79). Plasmid D N A was purified using the Quantum Prep kit (BioRad, Mississauga, ON). To construct pT7II9 and pT7II10, a 2,064 bp D N A fragment containing bphAE was amplified from pDB31-II9 and pDB31-II10, respectively, using oligonucleotides 20 II910F-NdeI and II910F-HindIII (Table 2.1) and Pwo polymerase according to the manufacturer's instructions (Roche, Laval , QC) . Thirty reaction cycles were performed as follows: 95°C for 45 s, 45°C for 40 s and 72°C for 2 min. The recovered amplicon was digested with N d e l / H i n d l l l and ligated to similarly digested pT7-7. The sequences of the final constructs were verified at the N A P S Service unit. To construct the pT7-6a derivatives, the 1347 bp bphAE fragments from pT7II9 and pT7II10 were digested with EcoRV/SacII and ligated into the corresponding sites of pT7-6a. 2. 4. Purification of BPDO components 2.4.1 Terminal Oxygenase (BphAE) The terminal oxygenases were purified anaerobically as previously described (48). Accordingly, all preparations were manipulated under an atmosphere of N 2 (< 2 ppm 0 2 ) using an MBraun Labmaster glovebox (Stratham, U S A ) . Chromatography was performed on an A K T A Explorer 100 (Amersham Pharmacia Biotech, Baie d'Urfe, QC) interfaced to the glovebox to minimize the oxygen content of the purification buffers and protein fractions (93). A l l buffers were prepared using water purified on a Barnstead NANOpure U V apparatus to a resistivity of greater than 17 MQ»cm. Buffers were sparged with N 2 and equilibrated in the glovebox for at least 24 h prior to use. The washed cell pellet from 4 to 8 L o f culture was resuspended in 40 mis of buffer A (25 m M H E P E S , p H 7.3, 10% glycerol) containing 0.01 mg/ml DNase (Boehringer Mannheim, Burlington, ON) and disrupted by successive passages through a cell homogenizer (Emulsiflex C-5, Avestin, Ottawa, ON) operated at 15,000 psi. The suspension was sparged with argon and the cell debris was removed by ultracentrifugation at 400,000 g for 45 minutes in gas-tight tubes. The clear supernatant fluid, referred to as "crude extract", was brought into the glovebox and filtered through a 0.45 jam filter. 21 TABLE 2.1. Plasmids, strains and oligonucleotides used in this study. Strain, plasmid or oligonucleotide Relevant phenotype/genotype properties Reference Strains E. coli DH5ct F", r", m + (39) E. coli C41[DE3] Mutant of E. coli B L 2 1 [ D E 3 ] ; F", r", m" (62) E. coli LE392 F , r , m (24) E. coli SGI3009 F", r", m" (47) (pREP4) Plasmids pT7-7 and pT7-6 T7 promoter, C o l E l origin, A p r (84) pT7-7AE3 pT7-7 carrying 6p/zAEB356 (48) pT7-6a pT7-6 carrying &P/ZAEFGBCLB400 (43) pDB31 T7 promoter, p i 5 A origin, A p r (16) pDB31-II9 pDB31 carrying bphAEuy (4) pDB31-II10 pDB31 carrying bphAEuio (4) P R K N M C IncPl replicon, lac promoter p R K N M C carrying i s c R S U A h s c B A f d x o r B cluster from (67, 89) pPAISC-1 Pseudomonas aeruginosa P A 0 1 , T c r (67, 89) pT7II9 pT7-7 carrying bphAEU9 This study pT7II10 pT7-7 carrying bphAEmo This study pT7II9a pT7-6 carrying bphAEngbphFGBCiBm This study pT7II10a pT7-6 carrying /3/?/*AEIHO6P/ZFGBCLB40O This study pT7B356a pT7-6 carrying bphAEB356bphFGBCui4oo This study p E B R E 1 2 p L E H P 2 0 carrying /3/?/ZFLB4OO [8] pQE31G pQE31 carrying 6p/zGB356 [9] Oligonucleotides (restriction sites underlined) 1191 OF-Ndel 5' -T A C G G A A C A T A T G A G T T C A G C A A T C A A A G A A G - 3 ' II910R-HindIII 5 ' - C A T G A A G C T T G T A C C C C C T A G A A G A A C T G C - 3 ' B356-BspHI 5 ' - P - T G T T T A A C T T T A A G A A G G A G A T A T A C T C A T G A G T T C G A C T A T G A A A G A T A C C - 3 ' 22 The crude extract was divided into equal portions, each of which was loaded onto a Source 15Q (Amersham Pharmacia Biotech) anion exchange column ( 2 X 9 cm) equilibrated with buffer A . The column was operated at a flow rate of 10 ml/min. Bound proteins were eluted using a linear gradient o f N a C l from 0 to 0.2 M over 10 column volumes. Fractions with absorbance at 323 and 455 nm, characteristic of the Rieske-type [2Fe-2S] center, were concentrated to 5 m l by ultrafiltration using a stirred cell equipped with a Y M 30 membrane (Amicon, Nepean, ON) . The preparation was brought to 5% saturation with ammonium sulfate and divided into equal portions. Each portion was loaded onto a Phenyl Sepharose (Amersham Pharmacia Biotech) column ( 1 X 9 cm) equilibrated with buffer PS (buffer A containing 5% ammonium sulfate). The column was operated at a flow rate of 1 ml/min. The oxygenases were eluted in a decreasing ammonium sulfate gradient (5% to 0% over 4 column volumes). Brown-coloured fractions were concentrated to 10-15 mg/ml by ultrafiltration, flash frozen as beads in liquid N2, and stored at -80 C. 2.4.2 Ferredoxin (BphF) His-tagged ferredoxin from Burkholderia sp. LB400 (ht-BphFLB40o) was anaerobically prepared using the QIAexpress system from Qiagen (24). Briefly, the cell pellet from 4 L of culture of E. coli LE392 containing the plasmid p E B R E 1 2 was resuspended in the lysis buffer (20 m M M O P S , 300 m M sodium chloride, 5 m M imidazole, 1 m M magnesium chloride, 1 m M calcium chloride, 200 units/ml DNase, and 1 unit/ml RNase, p H 8.0) and disrupted as described for the oxygenases. The cellular extract was then flushed with argon for 20 min and the cellular debris was removed by ultracentrifugation at 45,000 g for 45 min using sealed tubes. The supernatant was brought into the glovebox and loaded onto a N i 2 + chelating column (14 x 0.5 cm). The resin was washed with 20 m M M O P S , 300 m M N a C l , 20 m M imidazole, p H 8.0, to remove non-specifically bound contaminants. Ht-BphFLB4oo was eluted with 20 m M M O P S , 300 m M sodium chloride, 150 m M imidazole, p H 8.0. The protein was then concentrated to 15 mg/ml, buffer exchanged to remove imidazole, flash frozen as beads in liquid N2, and stored at -80 C. 2.4.3 Reductase (BphG) His-tagged reductase from C. testosteroni B356 (ht-BphGB356) was prepared using the QIAexpress system from Qiagen (47). Briefly, the cell pellet from 4 L of culture of E. coli SG13009 (pREP4) containing the p Q E 3 1 G plasmid was resuspended in lysis buffer 23 (50 m M Na-phosphate p H 7.8, 300 m M N a C l , 10% glycerol (w/v)) and disrupted as described for the oxygenases. The cellular debris was removed by ultracentrifugation at 45,000 g for 45 min using sealed tubes. The supernatant was loaded onto a N i chelating column and washed with lysis buffer and two wash buffers (lysis buffer adjusted to p H 6.0 and containing either 20 or 40 m M imidazole). Ht-BphGe356 was eluted using wash buffer containing 100 m M imidazole. Protein was concentrated to 10 mg/ml, buffer exchanged to remove imidazole, flash frozen as beads in liquid N 2 , and stored at -80 C. 2.5. Protein analysis S D S - P A G E was performed using a 12% resolving gel and a BioRad miniProtean II cell. Gels were stained with Coomassie Blue according to standard protocols (55). Protein concentrations were determined using the Bradford reagent (19), with bovine serum albumin as a standard. For the purified enzymes, concentrations were determined spectrophotometrically using the appropriate extinction coefficients (Table 2.2). The extinction coefficients for BphAEng and BphAEno at 455 nm were determined in this study. Briefly, the absorption of a series of dilutions of the oxygenase o f known concentrations was measured in triplicate and related to the extinction coefficient by Beer's law. Oxygenase concentration was based on sulphur content assuming 6 S atoms per hexamer. Sulphur content was determined colourimetrically using A^N-dimethyl-p-phenylenediamine and sodium sulphide as a standard (22). This value was used in the assays performed with purified enzymes. Table 2.2 Extinction coefficients of the purified BPDO components Protein X (nm) e ( mM^cm'1) Reference BphAE L B 4oo 455 8.3 (38) BphAE B 356 455 10.1 (47) BphAEn9 455 8.4 This study BphAEmo 455 9.6 This study BphFLB400 326 9 (24) BphGB356 450 11.8 (47) 24 2.6. Steady-state Kinetic measurements Enzymatic activity was measured by following the consumption o f O2 using a Clark-type polarographic O2 electrode (Yellow Springs Instruments Mode l 5301, Ye l low Springs, OH) (93). The activity assay was performed in a thermojacketted Cameron Instrument Co. model R C I respiration chamber (Port Aransas, U S A ) connected to a Lauda Model R M 6 circulating bath. Data were recorded every 0.1s and initial velocities were calculated from the slope of the progress curve for each consecutive 6 s interval. The standard activity assay was performed in a total volume of 1.4 m l of air-saturated 50 m M M E S , p H 6.0 (25°C). The reaction mixture contained 150 u M biphenyl, 320 u M N A D H , 3.6 u M ht-BphF L B 4oo, 1.8 u M ht-BphG B 356 and 0.6 u M B p h A E . The assay was initiated by adding the oxygenase after equilibrating the reaction mixture with all other components for 30 s. The reaction buffer and stock solutions used in the assay were prepared fresh daily. Stock solutions and protein samples were prepared anaerobically. The electrode was zeroed on the day of use by adding an excess of sodium hydrosulfite to the buffer in the reaction chamber. It was calibrated using standard concentrations of catechol and an excess of catechol 2,3-dioxygenase. Act ivi ty determinations were corrected for the consumption of O2 observed in the absence of oxygenase. One unit of enzyme activity is defined as the amount of enzyme required to consume 1 umol of 02/min under the described conditions. Apparent steady-state kinetic parameters for biphenyl were determined by measuring rates of oxygen uptake in the presence of concentrations o f biphenyl from 0.1 to 150 u M . The Michaelis-Menten equation was fitted to initial velocities determined at different substrate concentrations using the least-squares fitting and dynamic weighting options of L E O N O R A (23). 2.7. HPLC and GC-MS analyses H P L C analyses were performed using a Waters 2695 Separations Module equipped with a Waters 2996 Photodiode Array Detector and a C-18 Waters Nova-Pak column (3.9 X 150 mm) (Waters Limited, Mississauga, ON) . The instrument was operated at a flow rate of 1 ml/min. Biphenyls were eluted with a 20 m l gradient of 50% to 90% acetonitrile in H2O. Samples of 100 ul were injected and the amount o f biphenyl was determined from the area of absorbance peak at the appropriate wavelength using a standard curve. Standard curves for each biphenyl were established by determining the 25 peak areas of known amounts of the biphenyl. A l l standard curves had correlation factors higher than 0.97. A l l standard samples were treated in the same way as reaction mixtures to account for losses of biphenyl that may occur during sample manipulation. G C - M S was performed using an H P - 5 M S equipped with an Agilent column 19091S-433 (0.25 mm X 30 m X 0.25 um) (Agilent, Mississauga, ON) . The instrument was run at a flow rate of 53.5 ml/min and a pressure of 10.7 psi. Standard curves for each biphenyl were established by determining the peak areas of known amounts of the biphenyl. A l l standard curves had correlation factors higher than 0.99. 2.8. Coupling measurements Coupling experiments were carried out using 50 m M M E S , p H 6.0, 25°C, an excess of biphenyl or congener, 350 u M N A D H and the same concentrations of B P D O components used in the standard activity assay. Reactions were initiated by adding oxygenase and quenched 1-4 minutes later by adding acetonitrile (1:1 v/v). Oxygen consumption was monitored using the 0 2 electrode. The amount of hydrogen peroxide was estimated using catalase, 650 U of which was added to the reaction mixture at the time corresponding to the acetonitrile quench. The amount of oxygen detected by the oxygraph upon addition of catalase was taken to represent 50% o f the total hydrogen peroxide produced during biphenyl transformation. The consumption of biphenyl was determined by H P L C (section 2.7). 2.9. Depletion of PCB mixtures by purified BPDOs Depletion assays were performed in 12 m l glass vials sealed with teflon caps in a total volume of 1.0 m l of air-saturated 50 m M M E S , p H 6.0 (25°C). The reaction mixture contained the same concentrations of B P D O components as the standard oxygraph assay, 350 u M N A D H , and 10 u M each of 3,3 ' -diCl, 4 ,4 ' -d iCl , 2,6-diCl, 2,3,4'-triCl, 2,3',4-t r iCl , 2,4,4'-triCl, 3,3',5,5-tetraCl and 2,2',5,5'-tetraCl biphenyls (4). Reactions were initiated by adding oxygenase and quenched immediately or 20 minutes later (each enzyme retained at least 60% of their activity after 20 min; data not shown). After quenching, 2,2',4,4',6-pentaCl biphenyl was added as an internal standard and the reaction was extracted twice with hexane. The hexane fractions were pooled, dried over anhydrous sodium sulphate and analyzed for P C B congener content by G C - M S . 26 2.10. Whole-cell assays E. coli C41(DE3) cells freshly transformed with the isc plasmid and either pT7-6a, pT7II9a, or pT7II10a were grown at 37°C to mid-log phase, induced with 0.5 m M I P T G and further grown at 22°C to an OD600 of 1.0. Cells were then harvested, washed twice with 50 m M sodium phosphate, p H 7.5, supplemented with 1 g/1 glucose, and resuspended in the same buffer at an OD600 o f 2.0. One-ml portions of this suspension were distributed in 12-ml glass vials with teflon caps. Each vial received 10 u,M of each congener in the P C B mixture described in section 2.8. The reactions were shaken at 25°C at 225 rpm and stopped after 0, 3, and 18 hours by adding a drop o f 1 N HC1. The reactions were then frozen at -80°C. Assays were performed in duplicate. Controls contained C41(DE3) cells without any plasmid and were otherwise treated the same. A n internal standard, 2,2',4,4',6-pentaCl biphenyl, was added to a final concentration of 10 u M . The samples were extracted twice with 1 m l of hexane, pooled, dried over sodium sulphate and transferred to G C vials. The P C B content was analyzed as described in section 2.7. Protein levels in the different strains were verified using Sypro Ruby-stained denaturing gels (SDS polyacrylamide) of whole cells. Band intensities were quantified with ImageQuant 5.2 (Amersham Pharmacia). The levels of each of B p h A E F G were comparable in all assays. 2.11. Characterization of the transformation product of 2,4,4'-triCl biphenyl by BPDOB356 To characterize the BPDOe356-catalyzed transformation product of 2,4,4'-trichlorobiphenyl, two reactions were carried out in parallel. Each contained 600 u M of the substrate, 1.4 m M N A D H , 5 u M ht-bphG, 15 u M ht-bphF and 3 u M B p h A E . After 30 minutes, reactions were quenched with acetonitrile and the product was purified by H P L C and dried under a stream of nitrogen. The sample was resuspended in acetone-^6 and analyzed using a 500 M H z Varian N M R spectrometer (Department of Chemistry, U B C ) . 27 3. RESULTS 3.1. Purification and characterization of BPDO Relevant details o f the anaerobic purification of the oxygenase components o f B P D O are summarized in Table 3 . 1 . The enzymes were estimated to be greater than 9 0 % pure as judged by SDS polyacrylamide gel electrophoresis ( S D S - P A G E ) followed by Coomassie Blue staining and quantifying with ImageQuant 5 .2 (Amersham Pharmacia) (Fig. 3 .1 ) . This is comparable to previous results obtained with BPDOB356 purified both aerobically and anaerobically ( 4 4 , 4 8 ) . The ferredoxin and reductase components were estimated to be greater than 9 5 % pure (Fig.3.1) . Anaerobically purified BphAEL^oo and BphAEB356 had specific activities of 0 .2 and 4 U/mg, respectively. BphAEng and BphAEmo had specific activities of 0 .6 and 0 .3 U/mg, respectively. To ensure the optimum activity of the reconstituted B P D O , the electron-transfer activities o f BphFLB400 and BphGs356 were tested using the cytochrome c reduction assay ( 8 5 ) before using them with B p h A E (data not shown). a subunit, BphA 51 KDa Figure 3.1. SDS-PAGE of the purified components of BPDO. Lanes 1-4, BphAEB356, BphAELrwoo, BphAEi© and BphAEmo ( 1 5 ug each). Lane 5 , Broad Range prestained S D S - P A G E Standard (Biorad, Mississauga, O N ) . Lane 6 , BphFL B400 ( 1 2 kDa , 1 5 |ag). Lane 7 , B p h G B 3 5 6 ( 4 3 kDa, 1 5 ug). 2 8 Table 3.1. Purification of the oxygenases Enzyme/Purification step Total protein Total activity Specific activity Yield mg U U/mg % BphAELB4uo Crude extract 1069 32 0.03 (0.01) 100 Source Q 72 7 0.10(0.02) 22 Phenyl-sepharose 15 3 0.22 (0.03) 9 BphAE B356 Crude extract 1200 600 0.5 (0.1) 100 Source Q 153 230 1.5 (0.5) 38 Phenyl-sepharose 41 164 4.0 (0.3) 27 BphAEno Crude extract 2196 198 0.09 (0.03) 100 Source Q 105 32 0.3 (0.1) 16 Phenyl-sepharose 41 25 0.6 (0.1) 13 BphAEmo Crude extract 1314 40 0.03 (0.02) 100 Source Q 174 17 0.12(0.03) 43 Phenyl-sepharose 28 8 0.30 (0.02) 20 Activi ty units (U) are described in Materials and Methods. Standard deviations (n = 4) are indicated in parentheses. 29 The sulphur content of each oxygenase preparation ranged from 5.7 to 5.9 moles of S per ct3p3 hexamer. The iron content ranged from 8.1 to 9.6 moles per hexamer. These values indicate that the oxygenases contained full complements o f the Rieske-type [2Fe-2S] clusters and the mononuclear F e 2 + centres. Under aerobic conditions, the absorbance spectrum was characteristic of an oxidized Rieske-type center [2Fe-2S] cluster, with maxima at 323 and 455 nm and a shoulder at 575 nm (Figure 3.2). The ratio of the absorbance maxima at 280 nm and 323 nm ranged from 7 to 8.5. The extinction coefficients for ISPng and ISPmo were 8.4 and 9.6 mM" 1 cm" 1 , respectively (based on sulphur content). < 0-1 , , , r-310 410 510 610 710 Wavelength (nm) Figure 3.2. The UV-vis absorption spectrum of oxidized BphAEn9- The maxima at 323 and 455 nm, as well as the shoulder at 575 nm, are characteristic of the oxidized Rieske-type [2Fe-2S)] cluster. 3.2. Steady-state kinetic analysis and uncoupling constants for biphenyl utilization A l l four B P D O variants exhibited Michaelis-Menten kinetics for the dependence of the initial rate o f oxygen uptake on the concentration of biphenyl. To estimate the apparent steady-state kinetics parameters, the concentration of biphenyl was varied from 0.1 to 150 u M , which exceeds its solubility limit o f 45 u M . Representative data for BPDO356 and BPDO119 are shown in Figure 3.3. 30 B P D O B35<5 Biphenyl (pM) B P D O 119 Biphenyl (pM) Figure 3.3. Steady-state dihydroxylation of biphenyl by A ) BPDO356 and B) BPDO119. The graphic data shown here are from a single representative experiment. The solid line represents a fit of the data to the Michaelis-Menten equation using the least squares, dynamic weighting options of L E O N O R A . Similar results were obtained using different enzyme preparations. The fitted parameters for B P D O B356 averaged over 5 experiments were Km= 20 ± 4 u M and V m a x = 150 ± 10 p-M-min" 1. The averaged fitted parameters for BPDO119 (n = 5) were Km= 0.3 ± 0.2 u M and V m a x = 24 ± 2 nMmin" 1 . A l l experiments were performed using M E S p H 6.0, 25°C. 31 The apparent kinetic parameters for each o f the four enzymes are shown in Table 3.2. O f the four variants, the specificity constant (kcai/Km) o f BPDOLFMOO for biphenyl was highest, and was approximately 10-fold greater than that of B P D 0 B 3 56- Interestingly, B P D O B356 exhibited the highest turnover number, kcat, but its Km was a hundred times higher than that of BPDOLB400- With respect to these parameters, BPDO119 and BPDOmo fall between the two natural variants. However, BPDO119 is more similar to BPDOLB4OO while BPDOmo is more similar to BPDOB356-In each o f the four enzymes, the consumption of O2 was well coupled to the consumption of biphenyl (Table 3.2). That is, in the presence of a saturating concentration of biphenyl (150 uM), the amount of biphenyl consumed corresponded to the amount of O2 utilized within experimental error. Moreover, no hydrogen peroxide, a possible uncoupling product, was detected upon the addition o f catalase to reaction mixtures. Table 3.2. Kinetic parameters and coupling constants of BPDOB356? BPDOLB400> BPDO119 and BPDOmo using biphenyl as substrate. BPDO ^m(uM) kca/Km(\106 M 'Y 1 ) Biphenyl:02 H 20 2:0 2 B356 20 (4) 4.1 (0.2) 0.21 (0.04) 1.0 (0.2) N D LB400 0.18(0.03) 0.4 (0.1) 2.4 (0.7) 1.1 (0.1) N D 119 0.3 (0.2) 0.67 (0.08) 2(1) 1.0 (0.1) N D mo 2(1) 1.03 (0.05) 0.5 (0.3) 0.9 (0.2) N D Experiments were performed using 50 m M M E S p H 6, 25°C. The values of the parameters and their standard deviations (provided in parentheses) were calculated using the least squares and dynamic options of L E O N O R A (Cornish-Bowden 1995). Additional experimental details are provided in "Materials and Methods". N D , not detected. 32 3.3. The reactivity of BPDO variants with individual chlorinated biphenyls The reactivity of each B P D O with each of five different chlorinated biphenyls was examined over two minutes at a single congener concentration (50 uM) . Biphenyl depletion was followed by H P L C and the O2 depletion was followed with the oxygraph. The percent of depletion of the different substrates by each of the four enzymes is summarized in Table 3.3. O f the four isozymes, B P D O B 3 5 6 showed the best congener-transforming activity: it transformed all o f the five congeners at least as fast as any other isozyme did. O f particular note, B P D O B 3 5 6 depleted 2 ,2 ' -d iCl biphenyl just as well as BPDOLB400- B y contrast, BPDOmo had the poorest ability to transform congeners, significantly depleting only 2,3'- and 3,3-diCl biphenyls. The overall congener-depletion activity of BPDO119 was intermediate between that of the two parental enzymes, except for 4 ,4 ' -d iCl biphenyl, which BPDO119 transformed more slowly than either parental enzymes. Table 3.3. The depletion of individual chlorinated biphenyls by purified BPDOs % Depletion Congener BPDOB356 BPDOL B400 BPDO119 BPDOmo Biphenyl 100 48 100 100 3,3'-diCl 100 24 24 40 2,4,4'-triCl 82 <10 32 <10 2,2'-diCl 60 60 48 64 4,4'-diCl 36 16 <10 <10 2,6-diCl 24 <10 20 <10 Assays were performed using 50 m M M E S , p H 6, 25°C. Each substrate was tested individually using an initial concentration of 50 u M . The depletion of the substrates was followed by H P L C . Reactions were stopped after 2 min. A l l assays were performed in triplicate. The standard error was less than 10%. The rate o f depletion and the coupling constants were also determined for all substrates and enzymes (Table 3.4). None of the four enzymes transformed any of the tested congeners significantly faster than biphenyl. Except for biphenyl and, in the case 33 of BPDO119 and BPDOLB400, 2 ,2 ' -d iCl biphenyl, the consumption of the congener and O2 was uncoupled. In most cases, the O2 that did not react with the corresponding P C B was detected as H2C>2 in the reaction. However, not all uncoupling resulted in H2O2 production, suggesting that there is also some H2O production. Considering both the activities of the enzymes towards the different congeners and the uncoupling values, the apparent substrate preference of each enzyme at 25°C is as follows: B P D C W biphenyl > 3,3 ' -diCl > 2,4,4'-triCl > 4 ,4 ' -d iCl ~ 2 ,2 ' -d iCl > 2,6-diCl BPDOUMOO : biphenyl ~ 2 ,2 ' -d iCl > 3,3 '-diCl > 4 ,4 ' -d iCl > 2,4,4'-triCl ~ 2,6-diCl BPDO119: biphenyl > 2 ,2 ' -d iCl > 3,3 ' -diCl ~ 2,4,4'-triCl > 2,6-diCl > 4 ,4 ' -d iCl BPDOmo: biphenyl > 2 ,2 ' -d iCl > 3,3 ' -diCl 34 Table 3.4. The reactivities of purified BPDOs with individual congeners BPDOB356 BPDOLB40o BPDO„9 BPDO,„o Congener Act (nmol bph/min) Bph:0 2 H 2 0 2 : 0 2 Act (nmol bph/min) Bph:0 2 H 2 0 2 : 0 2 Act (nmol bph/min) Bph:0 2 H 2 0 2 : 0 2 Act (nmol bph/min) Bph:O z H 2 0 2 : 0 2 Biphenyl 55.3 (0.6) 1.0 (0.2) N D 12(4) 1.1(0.1) N D 44(2) 1.0 (0.1) N D 25(1) 0.9 (0.2) N D 3,3*-diCl 32.2 (0.2) 0.7 (0.2) 0.2(0.04) 6(2) 0.4 (0.3) 0.3 (0.1) 6(2) 0.26 (0.05) 0.31 (0.01) 10(1) 0.8 (0.03) 0.6 (0.3) 2,4,4'- triCl 20.5 (0.3) 1.0 (0.2) 0.31(0.02) 1.1 (0.3) 0.15(0.06) 0.7 (0.2) 8(1) 0.2 (0.1) 0.5 (0.1) N D N D N D 2,2'-diCl 15(1) 0.33 (0.03) 0.6(0.2) 15(4) 1.0(0.3) N D 12(2) 0.6 (0.2) N D 16(4) 1.2 (0.4) 0.4 (0.1) 4,4'-diCl 9(5) 0.4 (0.2) 0.4(0.2) 3.9 (0.3) 0.3 (0.1) 0.4 (0.2) 1 (0.4) 0.05 (0.01) 0.15(0.05) N D N D N D 2,6-diCl 6(1) 0.29 (0.05) 0.4(0.1) 1.5 (0.6) 0.12(0.04) 0.9 (0.1) 5(1) 0.6 (0.2) 0.4 (0.2) N D N D N D Assays were performed using 50 m M M E S p H 6, 25°C. Each substrate was tested individually, with a final concentration of 50 u M . The depletion of the substrates was followed by H P L C . The reaction times ranged from 1 to 4 minutes. The standard deviations for each value are indicated in parentheses. The amount of H2O2 detected upon addition of catalase was taken to be 50% of the total H2O2 produced during the reaction. 3.4. Characterization of the transformation product of 2,4,4'-triCl biphenyl by BPDOB356. A s shown in Table 3.3, B P D O B356 transformed 2,4,4'-triCl biphenyl at a relatively high rate. This was unexpected because previous studies had indicated that this enzyme had poor activity against double para-substituted congeners (1, 4). The metabolite produced by the hydroxylation of 2,4,4'-triCl biphenyl by B P D O B356 was purified by H P L C and absorbed maximally at X = 293.5 nm, which is within the range of Xmax o f other dihydrodihydroxydiols (3). Moreover, the incubation of 2,4,4'-triCl biphenyl with the first three purified enzymes of the pathway, B p h A E F G B C , yielded a H O P D A characterized by a bright yellow color. The color was not observed when the congener was incubated in the presence of purified B p h A E F G C (i.e., no dihydrodiol dehydrogenase), indicating that the BPDOa356-catalyzed dihydroxylation o f 2,4,4'-triCl biphenyl did not involve a dehalogenation. The purified metabolite of B p h A E was analyzed by N M R (Figure 3.4). The chemical shifts and coupling constants of the protons were consistent with the presence of four protons on the non-aromatic ring (excluding the hydroxyl protons), which are upfield of the aromatic ring due to the shielding effect of the - O H groups. The values are comparable to theoretical values predicted using ChemDraw Ultra 7.0 (CambridgeSoft, Cambridge, U S A ) as well as to the values obtained for similarly chlorinated 2,3-dihydroxylated compounds (7, 75). The transformation product was thus identified as 2,3-dihydro-2,3-dihydroxy-2',4,4'-triCl biphenyl (Figure 3.4), indicating that B P D O B356 catalyzes the 2,3-dihydroxylation of the congener on the monochlorinated ring. 36 ci Figure 3.4. The NMR spectrum of the product of 2,4,4'-trichlorobiphenyI dihydroxylation by BPDOB356. *H N M R (300 M H z , acetone-d6, 8): 4.5 (1H, d, J= 6 Hz , Hr/ H3>), 4.95 (1H, d, J= 6 Hz , H2>/H3>), 6.12 (1H, d, J= 6.1 Hz , H 5 V H 6 - ) , 6.43 (1H, d, J= 6.1 Hz , H 5 V H 6 -) , 7.65 (2H, H 3 / H 5 / H 6 ) , 7.5 (1H, H 3 / H 5 / H 6 ) . The data collected were not enough to assign protons 3, 5, 6, 5' and 6' to a specific chemical shift. The intensity of the peak for H 2 7 H 3 - was affected by the water signal suppression specified in the instrument. 3.5. The depletion of a PCB mixture by purified BPDOs The activities of the purified B P D O s were investigated using a mixture of 8 congeners described by Barriault et al. (4). This mixture contained 10 u M each of the following biphenyls: 3,3 ' -diCl , 4 ,4 ' -diCl , 2,6-diCl, 2,3,4'-triCl, 2,3' ,4-triCl, 2,4,4'-triCl, 3,3',5,5-tetraCl and 2,2',5,5'-tetraCl. The percentage depletion of each congener in the mixture after 20 min is summarized in Table 3.5. Consistent with the assays performed with individual congeners, B P D 0 B 3 5 6 showed the best congener-transforming activity: it was the only variant that significantly depleted all congeners and, with the exception of 2,3,4'-triCl and 2,2',5,5'-tetraCl biphenyls, transformed each congener faster than any of the other variants. Indeed, B P D O B 3 56 was the only variant that detectably depleted 2,6-diCl, 3 ,3 ' -diCl and 4,4 ' -diCl biphenyls. B y contrast, B P D O L B M O detectably depleted only 4 of the congeners in the mixture. However, it depleted 2,3,4'-triCl and 2,2',5,5'-tetraCl biphenyls faster than B P D O B 3 5 6- The activity of B P D O n 9 was similar to that of BPDO L B 4oo while BPDOmo had 37 the lowest overall depletion activity. Nevertheless, both BPDO119 and BPDOmo depleted 2,3,4'-triCl biphenyl more efficiently than either parental enzyme. Table 3.5. The depletion of a PCB mixture by purified BPDOs. % Depletion Congener BPDOB356 BPDOL B400 BPDO u 9 BPDOmo 2,6-diCl 21(11) <10 <10 <10 3,3'-diCl 29(11) <10 <10 <10 4,4'-diCl 13(5) <10 <10 <10 2,3',4-triCI 80 (14) 57(5) 35 (13) 18(11) 2,4,4*-triCl 21(5) <10 10(4) <10 2,3,4'-triCl 49 (5) 65 (18) 82 (9) 73 (23) 2,2*,5,5'-tetraCl 19(12) 51(13) 25 (12) 23 (14) 2,2',3,3'-tetraCl 18(12) 16 (4) 16(6) 10(3) Assays were performed using M E S p H 6, 25°C. The mixture contained the 8 listed congeners. The depletion of the biphenyl substrates was followed by G C - M S using 2,2',4,4',6-pentaCl biphenyl as an internal standard. Standard deviations are indicated in parentheses ( n > 4). 3.6. The depletion of a PCB mixture by whole cells The ability of B P D OLB400, BPDO119 and BPDOmo to deplete congeners in a mixture was also tested using whole cells. The bphAE genes were co-expresssed in E. coli C41(DE3) with 6/?/ZFGLB400 (43). Overall, the whole-cell assays mirror what was observed in the enzyme assays except that the levels of depletion were higher in the former (Table 3.5). Thus, the substrate preference of B P D O L B 4OO was 2,3',4-triCl > 2,2',5,5'-tetraCl ~ 2,2',3,3'-tetraCl > 3 ,3-d iCl ~ 2,3,4'-triCl > 2,4,4'-triCl > 4,4' d i C l > 2,6-diCl. A s in the purified enzyme assays, BPDO119 depleted most o f the tested congeners faster than BPDOL B4OO> and displayed a degradation pattern more similar to that of B P D O B356 in purified enzymes assays. Whole cells containing BPDOmo did not degrade any of the congeners 38 Table 3.6. The depletion of a PCB mixture by E. coli cells expressing BPDOs. % Depletion BPDOLB4oo B P D O I W BPDOmo Congener 3 hours 18 hours 3 hours 18 hours 18 hours 2,6-diCl <10 <10 39 100 <10 3,3'-diCl 26 79 38 100 <10 4,4'-diCl 11 18 39 100 <10 2,3',4-triCI 49 100 67 100 <10 2,4,4'-triCl 18 37 36 99 <10 2,3,4'-triCl 26 80 89 100 <10 2,2',5,5'-tetraCB 40 100 45 99 <10 2,2',3,3'-tetraCB 39 100 35 96 <10 Cells contained genes encoding the oxygenase (£»/?/zAELB4oo, bphAEug or bphAEmo) and /3P/JFGBCLB400- Cells were grown to an OD600 = 1.0, harvested and resuspended to an OD600 of 2.0 in 50 m M sodium phosphate, p H 7.5 supplemented with 1 g/L glucose. The depletion of the biphenyl substrates (10 u M each in assay) was monitored by G C - M S using 2,2',4,4',6-pentaCl biphenyl as an internal standard. The reaction time was 18 hours. Standard errors were less than 5% (n > 4). Results obtained for BPDOmo were the same at 3 and 6 hours. 39 4. DISCUSSION In the current study, highly active preparations of B P D O oxygenases were produced to better investigate the structural basis of their respective reactivities. Accordingly, BphAE L B 4oo, BphAEn9, BphAEmo and BphAE B 356 were each heterologously expressed in E. coli and purified anaerobically as described for BphAEe356 (1)- These proteins did not possess an N-terminal his-tag, which appears to reduce the stability of the oxygenase (45). Yields of B p h A E ranged from 5 to 10 mg of highly active protein per litre of cell culture (Table 3.1). The sulfur and iron contents of the preparations indicated that each contained full complements of their Rieske-type clusters and mononuclear Fe centers. Moreover, the absorbance spectra o f the oxygenases were characteristic of a Rieske-type cluster (Fig. 3.2). The yields of BphAEB 356 and BphAEL B4oo and their specific activities in reconstituted systems correspond to those reported by Agar et al. (1) and Master et al. (in preparation) using essentially identical protocols. Consistent with the observation that aerobic purification typically results in loss of iron and partial destruction of the Rieske center, the specific activities of the four anaerobically purified enzymes observed in this study were greater than those reported for aerobic preparations (0.09 U/mg and 2.4 U/mg for BphAE LB4oo and BphAEB356, respectively)(4, 38, 44). Most attempts to investigate steady-state kinetic parameters for different purified B P D O s have been based on H P L C assays that measure the depletion o f biphenyl or fluorimetric assays that follow either consumption of N A D H or production of H O P D A in coupled assays with BphB and BphC. For well-coupled systems, the use of an O2 electrode provides a reliable, continuous assay to follow the dihydroxylation of biphenyl directly. For substituted biphenyls, the use of an O2 electrode enables a measure of the degree of uncoupling between O2 and biphenyl consumption. In each of the four studied B P D O variants, 0 2 consumption was well coupled to biphenyl dihydroxylation, validating the conclusion that BPD0B356 is the most active enzyme using biphenyl as a substrate, followed by B P D O n 9 , BPDOmo, and B P D O L B4OO (Table 3.1). The higher specific activity of BPDOB356 over BPDOLB4OO has consistently been reported. However, preliminary results obtained with purified his-tagged BPDO119 and BPDOmo indicated a higher activity of both mutants over B P D 0 B 3 5 6 (4). The current results are more reliable as they were obtained using anaerobically purified preparations of native (i.e., non-tagged) proteins and a continuous assay. 40 Steady-state kinetic studies revealed that B P D OLB400 has the highest apparent specificity constant for biphenyl, followed by BPDO119, BPDOmo and B P D O B356 (Table 3.2). These results agree with those previously reported for the parental enzymes under identical conditions (1, 60). However, previous results obtained with his-tagged enzymes indicated that B P D OLB400 and B P D OB356 had similar Km values for biphenyl (46). This could reflect the influence of the his-tag or the lower sensitivity of the assay used. Interestingly, B P D O B356 is the only variant whose Vmax was observed at concentrations o f biphenyl that exceed the latter's solubility limit (45 uM) . Similar results were observed by Imbeault et al. for biphenyl as well as for 2,2'- and 3,3 ' -diCl biphenyls (48). The authors suggested a partitioning of the substrate from the solid phase to the active site. The kinetic parameters of mutant BPDO119 and BPDOmo for biphenyl are within the range of those of their parental enzymes. This is consistent with the observation that most engineered B P D O s described to date degrade biphenyl at slower rates than their parental enzymes (42, 87, 99). This likely reflects the optimization of the parental enzymes for their natural substrate. Interestingly, the identity of the residue at position 267 appears to have a much greater effect on the steady-state parameters for biphenyl than the identity of the residues at positions 335, 336, 338 and 341 combined (Table 3.2). The abilities of the four variant B P D O s to transform P C B congeners were investigated using three different methods: (1) purified enzymes and individual congeners; (2) purified enzymes and a mixture of 8 congeners; and (3) whole cells and a mixture o f 8 congeners. Overall, the three approaches yielded similar results although small differences were apparent. Thus, the rate of depletion of the individual congeners was lower in the presence of competing congeners, consistent with previous studies (12). Moreover, enzymes in whole cells transformed congeners faster than the reconstituted dioxygenases, consistent with previous reports (17, 37). It seems unlikely that these differences arise from a lower quality of the purified B P D O components versus those in the cell as the former where prepared anaerobically and were highly active. The differences may be due to factors such as different relative levels of the B P D O components and different uptake of the congeners by the cells. More particularly, the ratio of the expression of the ferredoxin, reductase and oxygenase in whole cells is likely to differ from their ratio in the assays with purified enzymes. In this study, the overall concentrations o f B p h A E were roughly the same in the whole cell assay (0.53 uM) and in the in vitro assay (0.6 uM) . However, the local concentration of B p h A E inside the cells was a thousand times higher (data not shown). The ratio B p h G : B p h A E ranged between 1.2 and 1.4 in the whole cell assay, but it was three times higher in the in vitro assay. In 41 general, it is difficult to compare results obtained by different researchers and to establish the substrate preference of B P D O s due to differences in biocatalyst preparation and how the assay was performed. The current study nevertheless demonstrates that it is possible to minimize such differences so that results can be compared. Moreover, although the data obtained with purified enzymes might be the most useful for mechanistic and biochemical studies, assays performed using whole cells provide valuable insights into the physiological behaviour of the enzyme. The relative preference of B P D OLB400 for the 13 congeners investigated in the current study is consistent with data reported by others (Tables 3.3, 3.5. and 3.6): biphenyl > 2 ,2 ' -d iCl > 2,3',4-triCl > 2,2',5,5'-tetraCl ~ 2,2',3,3'-tetraCl > 2,3,4'-triCl ~ 3,3 ' -diCl > 4 ,4 ' -d iCl > 2,4,4'-triCl ~ 2,6-diCl biphenyl. More specifically, other researchers have reported that the enzyme dihydroxylates biphenyl and 2,2 ' -diCl biphenyl at similar rates (32, 87). Moreover, the apparent preference o f purified B P D OLB400 for 2,2'- > 3,3'- > 4 ,4 ' -d iCl biphenyls agrees with previous reports using: (1) whole-cell assays with either individual congeners or a mix of the three (8); (2) whole-cell assays using more complex mixtures (3, 31, 66); and (3) purified enzyme with single congeners under the same conditions used in this study (1). Similarly, the enzyme transformed 2,3,4'- and 2,3',4-triCl biphenyls at similar rates (83, 99) in a process that does not involve dechlorination, as previously reported (6). In the current study, the depletion of 2,2',3,3'-tetraCl biphenyl was lower by the purified BPDOLB4OO than by whole cells. This may indicate that this congener is more rapidly taken up by cells than the others. Certainly, the ability of BPDOLB4OO to catalyze the 3,4-dihydroxylation of 2,2',5,5'-tetraCl biphenyl has been widely reported. In whole-cell assays, B. xenovorans LB400 depleted 2,2',5,5'-tetraCl and 2,2',3,3'-tetraCl biphenyls at similar rates (17, 37). B y contrast, E. coli cells expressing B P D OLB400 preferentially depleted 2,2',5,5'-tetraCl biphenyl (6). The transformation of 2,4,4'- t r iCl and 2,6-diCl biphenyls by purified B P D OLB400 has not been reported to date. Results obtained using whole-cell assays expressing B P D OLB400 and a mixture of congeners are ambiguous regarding the activity of the enzyme toward double ;?ara-substituted congeners, such as 4 ,4 ' -d iCl and 2,4,4'-triCl biphenyl. Some studies report a total lack of activity against such congeners (4, 6), others report a favoured attack of 2,4,4'-triCl over 4 ,4 ' -d iCl biphenyl when both are present in the reaction (17, 65), and yet others report similar values (20). However, the introduction of a second ortho-chloro substituent, to give 2,2',4,4'-tetraCl biphenyl, improves the 42 degradation o f this congener by BPDOLB4oo, in agreement with the enzyme's reported preference for double or^o-substituted congeners (20). The depletion of both 2,4,4'-triCl and 4 ,4 ' -d iCl biphenyls in presence of a mixture of congeners was comparable in this study, although depletion of the former by whole cells was slightly higher. Results obtained with whole cells of B. xenovorans LB400 in virtually identical conditions (Barriault et al., personal communication) also showed a higher depletion of 2,4,4'-triCl biphenyl, contributing to the ambiguity. Other than this, the results obtained with whole cells of B. xenovorans LB400 verified the degradation pattern observed in the current study. Overall, the current results substantiate and extend the conclusion that the introduction of ortho-cMoxo substituents to an otherwise meta- and/or para-QX biphenyl renders the congener more susceptible to attack by BPDOLB4OO despite the increased level of chlorination. A s discussed below, this is consistent with modeling experiments that indicate that non-planar congeners fit into the active site of BPDOLB400-B y contrast, the substrate preference of B P D OB356 differed from that of previous studies (Tables 3.2, 3.5 and 3.6): 2,3',4-triCI > 2,3,4'-triCl > biphenyl > 3,3 ' -diCl > 2,4,4'-triCl > 4 ,4 ' -d iCl ~ 2 ,2 ' -d iCl > 2,6-diCl > 2,2',3,3'-tetraCl ~ 2,2',5,5'-tetraCl. Moreover, in assays using purified components, B P D O B356 was more active than the other purified B P D O s against all tested congeners with the exception o f 2,2',5,5'-tetraCl biphenyl. The current study is consistent with previous studies reporting the apparent substrate preference of purified BPDO B356 for 3,3'- > 4,4'- > 2 ,2 ' -d iCl biphenyls (48). However, previous assays performed using whole-cell assays expressing His-tagged enzyme reported low activity of the enzyme against most congeners in the mixture, except for 2,3',4-, 2,3,4'-triCl and 3,3 ' -diCl biphenyls (4). While these three congeners were the most depleted by the purified enzyme in the current assay, every other congener was also depleted. The high activity of BPDO B356 against the 2,6-diCl and 2,4,4'-triCl biphenyls (Table 3.5) is of particular interest and has not been reported prior to the current study. The metabolite produced by the hydroxylation of 2,4,4'-triCl biphenyl by B P D 0 B356 was characterized as 2,3-dihydro-2,3-dihydroxy-2',4,4'-triCl biphenyl. The results were further confirmed by G C - M S using a His-tagged preparation of the enzyme (Barriault et al., personal communication). Studies with His-tagged preparations further confirmed that the activity of B P D O B 3 5 6 was much higher than that o f either B P D OLB400 or BPDOp4, a variant of BPDOLB4OO that degrades most congeners at a higher rate than BPDOLB400 (6). Although the sequence identity between BPDO B356 and each of BPDOLB4OO and B P D OKF707 is very high (75 and 76%, respectively), the reactivity of BPDO B356 appears to be closer to that of BPDOKF707. B P D OKF707 also shows a high level 43 of depletion for both 2,4,4'-triCl and 4 ,4 ' -d iCl biphenyls, although it preferentially degrades/?ara-substituted congeners over ort/zo-substituted congeners (20, 28, 37). Barriault et al. recently studied the products of the BPDO-catalyzed transformation of 2,6-diCl biphenyl (personal communication). Each of B P D OB356, B P D OLB400, BPDO119 and BPDOp4 catalyzed the formation of two hydroxylation products, both of which were dichlorinated. This implies that one of these products is 3,4-dihydroxylated. Quantification revealed that B P D OB356 produced higher levels o f the two products than any of the other variants. This is the first report o f 3,4-dihydroxylation by BPDOB356- B P D O B 3 56 has not been as wel l studied as B P D OLB400 or BPDOKF707-Moreover, most the previous studies have been conducted using whole cells, usually expressing His-tagged enzyme and using different mixtures of congeners (5, 8, 44, 45). Clearly, these approaches have limited what we have learned about this enzyme. A particularly striking characteristic of BPDO B356 revealed by this study is the high rate at which it transforms 2,3',4-triCl and 2,3,4'-triCl biphenyls (Table 3.5). It would be interesting to investigate the coupling o f O2 and congener consumption in these reactions, as well as to elucidate the transformation products. BPDOLB4OO transforms 2,3' ,4-triCl biphenyl to two products with a ratio of 1:4, resulting from the 2,3-dihydroxylation of the respective rings (83). In principle, B P D O B356 could also catalyze the hydroxylation of both rings. However, the preference of the enzyme for meta-substituted congeners may favour an attack in the least chlorinated ring. A s for 2,3,4'-t r iCl biphenyl, which is also degraded to almost twice the level of any other congener, attack probably proceeds via 2,3-dihydroxylation of the least chlorinated ring. The 2,4-substitution pattern of 2,3',4-triCl biphenyl is also present in 2,4,4'-triCl biphenyl, which the enzyme 2,3-dihydroxylated at a high rate in single congener assays (Fig. 3.4). The preference of B P D O B356 for 2,3',4-triCl biphenyl over 2,4,4'-triCl biphenyl is consistent with its preference of 3,3 ' -diCl over 4 ,4 ' -d iCl biphenyl. That is, congeners containing a meta-substituent on the attacked ring appear to better substrates, presumably due to a better fit in the active site. Another interesting characteristic of B P D O B356 is that it transforms both 2,2',5,5'-and 2,2',3,3'-tetraCl biphenyls at a rate similar to that of most d i C l biphenyls tested (Table 3.5). Neither B P D O B 3 56 nor B P D OKF707 have been reported to transform either 2,2',5,5'- or 2,2',3,3'-tetraCl biphenyls, even in assays using purified enzymes (5, 28). In recent whole cell assays performed under essentially identical conditions to those of the current study, Barriault et al. confirmed the high activity of B P D O B356 on these 44 "recalcitrant" congeners (data not shown). Further research is being conducted to determine the activity of E. coli cells expressing BPDOB356-The overall substrate preference of BPDO119 was closer to that of B P D O L B W O than that of BPDO B356 (Tables 3.3, 3.5 and 3.6): biphenyl > 2 ,2 ' -d iCl > 2,3,4'-triCl > 2,3'4-t r iCl > 2,2',5,5'-tetraCl > 2,2',3,3'-tetraCl > 3,3 ' -diCl ~ 2,4,4'-triCl > 2,6-diCl > 4,4'-d iC l . This is consistent with the steady-state parameters for biphenyl, which were also closer to those of BPDOLB4OO - Although BPDO119 was selected as a variant with superior PCB-transforming properties, the purified enzyme did not show superior activity against any individual congeners when compared to purified BPDOB356- When tested using a mixture of congeners, purified BPDO119 transformed only 2,3,4'-triCl biphenyl faster than either parental enzyme. The current whole cell assays are nevertheless consistent with previous whole-cell studies in that BPDOng-containing cells depleted most congeners faster than BPDOLB400-conta in ing cells. The apparent substrate preference of the enzyme in whole cell assays was thus similar to that observed previously (4). BPDOmo was the least active enzyme in this study. Nevertheless, in the mixed congener assay, the overall pattern of congener degradation by the purified enzyme was similar to that of BPDO L B 4oo and BPDO119 (Table 3.5). Interestingly, BPDOmo showed a slightly improved ability to degrade 3,3 ' -diCl biphenyl when compared to both B P D OLB400 and BPDO119 (Table 3.4). This could indicate an important role of Ala267 in the binding of meta-substituted congeners. On the other hand, the improved degradation of 2,4,4'-triCl and 2,6-diCl biphenyls by BPDO119 when compared to BPDO L B 40o was not observed in BPDOmo, suggesting again a crucial role of Ala267 in substrate binding. Finally, it is not clear why whole cells expressing BPDOmo did not degrade any of the congeners, although this is consistent with previously observations (4). The coupling between the consumption of O2 and the aromatic substrate reflects the ability of B P D O to utilize O2. According to the proposed mechanism for N D O (Fig. 1.6.), binding o f the aromatic substrate transforms the hexacoordinate active site Fe(II) to a pentacoordinate state. The pentacoordinate Fe(II) binds O2, which is then reduced yielding an activated oxygen species that reacts with the aromatic substrate. The efficiency of this reaction depends on the proximity between the two reactants in the active site and the exclusion of solvent molecules from this environment. B y analogy to what has been observed in cytochrome P450 monooxygenases (74), P C B s that do not occupy the active site o f B P D O in the same manner as biphenyl may lead to uncoupling in at least three situations: (1) the aromatic substrate is positioned too far from the 45 activated oxygen species; (2) the P C B does not displace key solvent species from the active site; or (3) the chloro substituents may occlude the attachment site o f the activated oxygen intermediate (48). Reaction of solvent species with the activated oxygen species results in uncoupling as depicted in Figure 1.7. In this study, the transformation of P C B s was uncoupled from the consumption of O2 in all but one case: the transformation of 2 ,2 ' -d iCl biphenyl by B P D OLB400 (Table 3.4). This uncoupling is consistent with the fact that P C B s are not the optimal substrates for these enzymes. For the wi ld type enzymes and BPDO119, the degree of uncoupling was inversely related to the rate of depletion, indicating a better fit o f these congeners into the active site. Results obtained with both B P D OLB400 and B P D O B356 and 2,2'-,3,3'-, and 4,4 ' -d iCl biphenyl are consistent with previous results obtained in identical conditions (1, 60). In the case of BPDO119, the rates of depletion for 2,6-diCl, 3 ,3 ' -diCl and 2,4,4'-triCl were comparable (within experimental error). The lower degree of uncoupling in the presence of 3,3 ' -diCl biphenyl indicates a better fit of the congener into the active site. Conversely, the high level of uncoupling observed in the presence of poorly degraded congeners indicates that these compounds bind relatively well to the enzyme. Such is the case for 2,6-diCl and 2,4,4'-triCl biphenyls for BPDOLB4OO , in which the rate of depletion is under 2 nmol/min but the ratio F ^ C ^ C h is close to one. Similarly, the lower uncoupling observed in the presence of these congeners for BPDO119 indicates that they fit the active site of BPDO119 better, and not that the enzyme has a higher affinity for them. Interestingly, the ratio Bph:C>2 was comparable between 2,2'- and 2,6-diCl, even though the depletion of the former was twice that of the latter. Not all uncoupling resulted in H2O2 production, suggesting that there is also some H2O production, although that is certainly minimal. The low activities of BPDOmo did not allow for a thorough investigation of the uncoupling. While it is difficult to assign specific roles to individual residues in determining congener preference, consideration of the crystal structures o f BphAEB356, BphAE LB400 and BphAEiig (Kumar and Bol in , personal communication), each free and in the presence of bound biphenyl, provides useful insights into the current data. The substrate-binding pocket may be divided into the proximal and distal regions, corresponding to the portions of the pocket that bind the hydroxylated and non-hydroxylated rings of biphenyl, respectively (Fig. 4.1.). In BphAE LB400, the residues that line the proximal portion of the pocket are Gln226, Phe227, Asp230, Met231, His323, Pro334, Asn337, Asn338, Ser379 and Glu385 (Fig. 4.1.). Residues lining the distal portion of the pocket include Thr 237, 46 Ile243, Tyr277, Ser283, Val287, Met319, Val320, Thr335, Phe336, As377, Phe378, and Phe384. O f these, Ile243, Ser283, Ala286, Val287, Val320 and Phe336 are of particular interest as they shift upon the binding of biphenyl to BphAELB400 (Fig. 4.2.). The equivalent residues in B p h A E B 3 5 6 are Val243, Ile283, Ser286, Val287, Phe320 and Asn336. Sequence alignments indicate that Ile243 and Val287 are well conserved among the oxygenase a subunits of BPDOB356, BPDOLB4OO , BPDOKF707, B P D O R H A I , and CDOJS375 (data not shown). Moreover, residues 283 and 336 are Leu/Ile and He in all o f these dioxygenases except BphAELB400- Residue 320 is not conserved at al l , and residue 286 is conserved in all but BphAEB356- Two other residues that shift significantly upon the binding of biphenyl in BphAELB400 are Val287 and Phe387. Figure 4.1. The substrate-binding pocket of BphAELB4oo- Ligands to the iron are shown in grey. Other residue in the pocket is shown in violet. Bound biphenyl is shown in pink, and the mononuclear iron is depicted as a purple sphere. 47 Figure 4.2. Shifts in active site residues of BphAELB4OO induced by biphenyl-binding. Residues of the substrate-free and biphenyl bound enzymes are shown in blue and brown, respectively. Only the residues that line the substrate-binding pocket and that shift significantly upon substrate binding are depicted. Bound biphenyl is shown in violet and the mononuclear iron is depicted as a purple sphere. Based on previous studies, residues known to play a role in determining congener preference include residues of regions I (237-238), II (278), III (335-341) and IV (377), identified by Mondello (Fig. 4.3.). Residue 237 from region I is located in the distal portion of the binding pocket (Fig. 4.1.), whereas residue 238 is located in the proximal portion. However, the latter is adjacent to His239, one of the ligands to the iron, and could explain its effect on the enzyme activity. Residue 278 (region II) is not part of the binding pocket, but is adjacent to 277, which makes up part of the distal portion of the pocket. Residues of regions III (335, 336 and 338) and IV (377) are located in the upper part of the binding pocket, either distal or proximal. The latter interacts with Phe387 through a hydrogen bond, and could explain the effect of Asn377 on the enzyme's activity. More particularly, Asn377 occurs in enzymes possessing broad substrate 48 specificity, such as B P D O L B 4 0 0 - B y contrast, this residue is Thr in dioxygenases possessing a narrow substrate specificity (66). Figure 4.3. A topology map of BphAs356 depicting the relative positions of the determinants of substrate specificity. Residues mutated in variants BphAng and BphAmo are shown as filled red squares. Residues in the first sphere o f the substrate-binding pocket are shown as open red boxes (residues 226, 227, 231, 243, 277, 283, 287, 323, and 336). Regions I, II, III, and IV defined by Mondello et al. (66) are labeled with green Roman numerals. The residues that were substituted in BphAEng and BphAEmo occur in two distinct parts of the enzyme: residues 335, 336, 338 and 341 (LB400 numbering) contribute to the substrate-binding pocket as mentioned above whereas residue 267 is remote from the active site (Figure 4.4.). Although the latter is close in sequence to region II identified by Mondello (residue 277), it is located on the upper surface o f the tnmer, approximately 21 A away from the mononuclear iron o f the active site. Due to its remote location, it is not obvious why this residue has such a large influence on the 49 reactivity o f B p h A E with biphenyl and PCBs . In BphAELBAOO , Ala267 forms a hydrogen bond with His272. These two residues are located on beta strands 15 and 16, respectively, which move slightly upon substrate binding. This movement might facilitate the binding of substrates by producing more volume in the active site. Replacement o f A l a by Ser may create a new arrangement in that region by changing the interactions among residues. The different activities o f BPDOmo and B P D O B 3 5 6 , two enzymes that both have Ser267, highlight the importance of sequence context on the influence of a specific residue. Figure 4.4. The substituted residues in BphAII9 and BphAEmo. The residues that are substituted in B p h A i ^ with respect to BphALB4oo are shown in blue (T335G, F336I, N338T, I341T) and the residue substituted in BphAEmo with respect to BphAELB400 is shown in red (A267S). The figure depicts the alpha subunit only. The C-terminal domain is brown, and the N-terminal, Rieske domain, at the back of the molecule, is blue. Bound biphenyl is shown in green, irons are depicted as purple spheres and sulfurs o f the Rieske cluster are represented as orange spheres. 50 Residues 335, 336, 338 and 341 form part of the roof o f the substrate-binding pocket and are also part of region III identified by Mondello, as discussed above. The sequence of region III, TFNNTRI, is shared by BphAE LB400 and BphAEHsso, from A. eutrophus H850 (66), two of the most versatile enzymes described in the literature. The corresponding sequence in BphAEe356 and B p h A E R H A i is G I N T I R T , and a similar sequence is also present in BphAE]CF707 and C D O . In the structure of the BphAE L B4oo :biphenyl complex, Phe336 is 4.3 A from C4 of the distal ring of the bound biphenyl, while residues 338 and 341 are - 10 A from the bound substrate. Residues from region III undergo small conformational changes upon binding o f biphenyl, mostly through their sidechains (Fig. 4.2.). This, together with the close p r o x i m i t y of Phe336 to the bound b ipheny l , indicates that these residues likely play an important role in positioning the substrate in the active site, probably in part through aromatic interactions. Consistent with its close location to C4 of distal biphenyl ring, replacement of Phe336 by a smaller residue, such as He as occurs in BphAEB356 or B p h A E r o , appears to enable a better fit o f 4,4'-substituted congeners, especially when combined with replacement of Thr335. This observation is supported in this study by the higher levels of transformation of 4 ,4 ' -d iCl and 2,4,4'-triCl by B P D O n 9 when compared to B P D O L B 4 0 0 and by the lower level of uncoupling with both congeners for BPDO119. The effect of Phe336 on substrate preference has been widely reported and suggests a close interaction o f these residues with the substrate (20, 66). Replacement of Ile335 in BphAEicF707 by the corresponding Phe336 from BphAE LB4oo did not only prevent the enzyme from hydroxylating 4 ,4 ' -d iCl biphenyl, but also induced the 3,4-dihydroxylation of congeners with a 2,5-substituted ring, a characteristic of B P D O L B 4 0 0 (87). Barriault et al. recently reported similar effects on regiospecifity toward 2 ,2 ' -d iCl biphenyl (6, 7). Replacement of Thr335 and Phe336 by A l a and Met/l ie resulted in an increase in 3,4-dihydroxylation, which was also affected in part by changes in Ile341. The crystal structure of B P D O L B 4 O O suggests a more subtle effect of mutations in positions 338 and 341 than in positions 335 and 336 (Fig. 4.4.). The presence of Thr at positions 338 and 341 has, however, been associated to a negative influence in the dihydroxylation of ort/20-substituted congeners (20, 54). In this study, the levels of depletion of 2 ,2 ' -d iCl biphenyl by B P D O B 3 5 6 and BPDO119 were comparable to those of B P D O L B 4 O O - B P D O B 3 5 6 and BPDO119 contain a Thr in positions 338 and 341, whereas B P D O L B 4 O O contains an Asn and He, respectively. The current results are thus more consistent with the structural data than previous studies. The replacement of region III in BphAELB4oo by that of BphAEKF707 produced a dioxygenase that combined the broad 51 substrate specificity of B P D O L B W O with the ability of B P D O K F 7 0 7 to degrade 4,4'-substituted congeners (28). These observations partially agree with the results obtained with BPDO119 and BPDOmo, indicating the effect of other amino acids in the overall reactivity of the enzyme. It should also be noted that a given mutation could have different effects on the structure/activity of an enzyme when combined with others. A s stated earlier, the combination of the additional mutation in bphAEmo and the mutations in region III seemed to affect the enzyme reactivity to a greater extent than the latter alone. The role o f the region III residues is hard to determine based on the results presented in this study. The only way to definitively establish the role of particular residues is by determining the crystal structure of the variant protein. The superposition of the active sites of B P D O L B 4 O O , B P D O B 3 5 6 and BPDOng reveals that the substrate-binding pocket of BPDO119 is slightly larger. Although little structural data of the relevant enzyme:congener complexes exists at this time, docking and minimization results indicate that B P D O B 3 5 6 readily accommodates 3,3-' and 2,2'-d i C l biphenyls in its active site in a catalytically competent configuration. B y contrast, when 2,2,',5,5'-tetraCl and 2,4,4'-triCl biphenyl are docked to B p h A E , the congeners sterically clash with residues 283, 286 and 287. However, it may be possible that Ile283 shifts slightly when 2,4,4'-triCl biphenyl binds, allowing for a better fit o f the substrate. A l l three enzymes accommodate 2,6-diCl biphenyl in a similar fashion, reflecting the existence of other factors on the activity of the enzymes against the congener. On the other hand, B P D O L B 4 O O can accommodate 3,3'- and 2 ,2 ' -d iCl biphenyls, as well as 2,2,',5,5'-tetraCl biphenyl, but there are also steric clashes with 2,4,4'-triCl biphenyl. Interestingly, BPDO119 is able to accommodate all of these congeners in a catalytically competent configuration, despite the lower congener-transforming of this variant with respect to B P D O B 3 5 6 - Based on docking and minimization experiments, BPDO119 binds 2 ,2 ' -d iCl and 2,2',5,5'-tetraCl biphenyl in a similar fashion to that of B P D O L B 4 O O , whereas binding of 3,3 ' -diCl biphenyl is similar to that of B P D 0 B 3 5 6 - Thus, BPDO119 is probably capable of catalyzing the 3,4-dihydroxylation of 2,2',5,5'-tetraCl and 2,2 ' -diCl biphenyls. The docking experiments further suggest that BPDO119 could catalyze the dechlorination on 2 ,2 ' -d iCl biphenyl. It is clear that the orientation of the substrate in the active site is strongly influenced by its chlorination pattern and the interactions of the chlorines with specific residues of the enzyme. Accordingly, a given amino acid substitution w i l l have different effects on the regiospecificity of different congeners. 52 Considerable effort has been invested in engineering B P D O in the last years. Most of this effort involved, however, mutations of residues in region III. Although these play an important role in the substrate preference of the enzyme, it is possible that some other, as yet unidentified, residues play an equally important role. The publication of the crystal structures of BphAERHAi and other ring-hydroxylating dioxygenases should not only help to identify some of these residues but should also provide a better understanding o f the role of the previously identified residues and the enzyme's mechanism. The upcoming publication of the crystal structures of BphAE B 356 , BphAELB4oo and BphAEng is of particular interest since they were obtained from highly active enzymes. These are also the first structures obtained from an engineered version of B P D O , and comparison with the wi ld type enzyme should allow for the thorough investigation of the role of the mutated residues. Similarly, most protein engineering efforts have involved the use of BPDO L B400 and BPDOKF707- The high PCB-transforming activity o f B P D O B 3 5 6 highlights the importance of including this and other less well characterized enzymes in engineering efforts, particularly directed evolution. This is particularly true for engineering B P D O for green chemistry, a venue that has yet to be fully exploited despite the studies that demonstrate that it is easy to modify the regiospecificity of the enzyme. The engineered versions of B P D O are rarely tested against other aromatic substrates. Such testing may reveal some interesting novel activities of the enzyme. 53 5. BIBLIOGRAPHY 1. Agar, N . Y. R. 2002. Identification of molecular determinants o f substrate specificity and activity for biphenyl dioxygenase from Comamonas testosteroni B-356. Concordia University, Montreal, Canada. 2. Ahmad, D., R. Masse, and M. Sylvestre. 1990. Cloning and expression of genes involved in 4-chlorobiphenyl transformation by Pseudomonas testosteroni: homology to polychlorobiphenyl-degrading genes in other bacteria. Gene 86:53-61. 3. Arnett, C. M., J. V. Parales, and J. D. Haddock. 2000. Influence of chlorine substituents on rates o f oxidation of chlorinated biphenyls by the biphenyl dioxygenase of Burkholderia sp. strain LB400 . A p p l Environ Microbio l 66:2928-33. 4. Barriault, D., M. M. Plante, and M. Sylvestre. 2002. Family shuffling of a targeted bphA region to engineer biphenyl dioxygenase. J Bacteriol 184:3794-800. 5. Barriault, D., C. Simard, H. Chatel, and M. Sylvestre. 2001. 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