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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 OF THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Microbiology and Immunology)  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A November 2005 © Leticia Gomez G i l , 2005  ABSTRACT Biphenyl dioxygenase ( B P D O ) is the first enzyme i n the bph pathway, catalyzing the dihydroxylation o f biphenyl and some polychlorinated biphenyls. The B P D O s o f different bacterial strains possess different abilities to transform P C B s . To better understand the molecular basis o f these different abilities, highly active preparations o f four  BPDOs  were  anaerobically purified  Burkholderia xenovorans L B 4 0 0 , two  and from  BPDOB356  characterized:  BPDOLB4OO  from  Comamonas testosteroni B-356, and  engineered variants, BPDOng and BPDOmo. BPDO119 is a variant o f BPDOLB400  containing four substituted residues: T335G, F336I, N338T, and I341T. These residues correspond to those found i n BPDOB356, belong to region III identified by Mondello  et  al, and contribute to the substrate-binding pocket o f the enzyme. B P D O m o contains these substitutions as well as A 2 6 7 S . Steady-state kinetics assays demonstrated that o f the four variants, BPDOB356 had the highest apparent & than that o f BPDOLB400-  cat  for biphenyl, which was 10-fold higher  B y contrast, BPDOLB4OO had the highest apparent substrate  specificity for biphenyl and was 10-fold higher than that o f BPDOB356- The steady-state parameters o f BPDO119 and B P D O m o for biphenyl were intermediate between those o f the two parental enzymes. The identity o f the residue at position 267 had a greater effect on the parameters than the identity o f the region III residues. In all variants, the consumption of oxygen was well-coupled to that o f biphenyl. The abilities o f 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 o f 8 congeners; and (3) whole cells and a mixture o f 8 congeners. The results obtained by each method were consistent. Most strikingly, BPDOB356 transformed a greater number o f congeners at a faster rate than the other enzymes. Previously unrecognized activities o f BPDOB356 include the 2,3-dihydroxyation o f 2,4,4'-triCl biphenyl as well as the 2,3- and 3,4dihydroxyation o f 2,6-diCl biphenyl. For B P D O B 4 0 0 and B P D 0 3 5 6 , the degree o f L  B  uncoupling was inversely related to how well the congener was transformed.  The P C B -  transforming abilities o f BPDO119 and B P D O m o were more similar to those o f BPDOLB4OO-  However, both showed improved ability to transform either para- (BPDO119)  or weta-substituted congeners (BPDOmo). The crystal structures o f  BPDO B4oo L  and  BPDO119 further confirmed the role o f residues i n region III i n the range o f substrates accepted by B P D O .  11  TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS  ii .  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 o f P C B s : the bph pathway  2  1.2.1 The bph operon 1.3 Dioxygenases  5 6  1.3.1 Ring-hydroxylating dioxygenases  6  1.3.2 Classification o f ring-hydroxylating oxygenases  7  1.3.3 Proposed mechanism for ring-hydroxylating oxygenases  8  1.3.4 Coupling between substrate consumption and dihydroxylation 1.4 Biphenyl dioxygenase ( B P D O )  10 11  1.4.1. Crystal structure o f B P D O  12  1.4.2. Substrate preference o f B P D O  15  1.4.3. B P D O engineering  16  1.5 A i m o f 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 o f 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  iii  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 o f P C B mixtures by purified B P D O s  26  2.10. Whole-cell assays  27  2.11. Characterization o f the 2,4,4' P C B degradation product  27  3. RESULTS  28  3.1. Purification and characterization o f B P D O  28  3.2. Steady-state kinetic analysis and uncoupling constants for the dihydroxylation o f biphenyl  30  3.3. The reactivity o f 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 BPDO 356  36  3.5. The depletion o f 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  B  iv  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 BPDO119 and BPDOmo using biphenyl as substrate  BPDOB356> BPDOLB4OO,  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 bphAEuv  or bphAEmo and Z?P/?FGBCLB4OO  £>P/JAELB4OO,  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 o f uncoupling i n ring-hydroxylating dioxygenases  10  Figure 1.8. Dihydroxylation o f biphenyl b y B P D O  11  Figure 1.9. The structural fold o f  BPDO 56  13  Figure 1.10. The metallocentres o f  BphAE 356  15  B3  B  Figure 3.1. S D S - P A G E o f the purified components o f B P D O  28  Figure 3.2. The U V - v i s absorption spectrum o f oxidized bphAEng  30  Figure 3.3. Steady-state dihydroxylation o f biphenyl by A ) BPDO356 and B ) BPDO119  31  Figure 3.4 . The N M R spectra o f the product o f 2,4,4'-trichlorobiphenyl dihydroxylation b y B P D O B 3 5 6  37  Figure 4.1. The substrate-binding pocket o f BphAELrwoo  47  Figure 4.2. Shifts in active site residues o f BphAE 4oo induced by biphenyl-binding. 48 LB  Figure 4.3. A topology map o f  BphA 356 depicting the relative positions o f the B  determinants o f substrate specificity  49  Figure 4.4. The substituted residues in BphAEng and BphAEmo  50  vi  ABBREVIATIONS ATP  Adenosine triphosphate  BPDO  Biphenyl dioxygenase  BPDOLFMOO  Biphenyl dioxygenase from  Burkholderia xenovorans L B 4 0 0  BPDOB356  Biphenyl dioxygenase from  Comamonas testosteroni B356  BPDORHAI  Biphenyl dioxygenase from  Rhodococcus sp. strain R H A 1  BPDOKF707  Biphenyl dioxygenase from  Pseudomonas pseudoalcaligenes K F 7 0 7  BPDO119  Engineered biphenyl dioxygenase  BPDOmo  Engineered biphenyl dioxygenase  BPH  Biphenyl  CDO  Cumene dioxygenase  CDOJS375  Cumene dioxygenase  DNTP  Deoxynucleoside triphosphate  DTT  Dithiothreitol  GC-MS  Gas chromatography-mass spectrometry  HPLC  H i g h performance liquid chromatography  EPTG  Isopropyl-beta-D-thiogalactopyranoside  ISC  Iron sulfur cluster assembly  MES  2-(N-morpholino)ethanesulfonic acid  NADH  Nicotinamide adenine dinucleotide  NBDO  Nitrobenzene dioxygenase  NBDOJS765  Nitrobenzene dioxygenase from  NDO  Naphthalene dioxygenase  NDO98164  Naphthalene dioxygenase from  NMR  Nuclear magnetic resonance  PAGE  Polyacrylamide gel electrophoresis  PCB  Polychlorinated biphenyl  Pseudomonas fluorescens IP01  Comamonas strain JS765  Pseudomonas strain N C T B 9816-4  vii  PCR  Polymerase chain reaction  UV  Ultraviolet  ACKNOWLEDGEMENTS I would like to thank m y supervisor, Lindsay Eltis, for his trust and patience. Thanks for allowing me to do things " m y way". Thanks to all past and present members o f the Eltis lab for their help and frienship. I must especially thank Pascal Fortin and Geoff Horsman, for their valuable advice and comments on m y research, and Sachi Okamoto and Thomas Heuser, for their constant support. M a n y thanks to m y committee members, T o m Beatty and B i l l M o h n , for looking after me and for reviewing m y thesis. I would also like to thank Diane Barriault and M i c h e l Sylvestre for allowing me to use their engineered enzymes and for their collaboration on some parts o f this work. M a n y thanks to Pravindra Kumar and Jeffrey B o l i n for providing such good quality crystal structures o f 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 . O n 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 m i familia, en especial a m i 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. S i n 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 o f biphenyl. The production process results in up to 209 congeners, each differing in position and number o f chlorine substitutions. Their nomenclature is based on the position o f these substitutions on the phenyl ring  (meta,  ortho, para) or according to the numbering o f 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 o f 60 to 90 congeners, such as Aroclor. The mixtures differ i n the extent o f chlorination and specific congener composition. Due to their nature, P C B s persist in the environment and accumulate i n the food chain, causing adverse health effects i n humans, such as liver and neuronal damage, alterations in the immune system and increased incidence o f 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  Cl  n {10-n} H  Para  Para  6'  5'  Polychlorinated Biphenyl (PCB) 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 o f 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) o f electron donors coupled to the utilization (reduction) o f electron acceptors. H i g h l y chlorinated P C B s act as electron acceptors because o f their high oxidation state and chlorines are then replaced by hydrogen (63). This is a highly selective process and none o f the microbial dechlorination patterns characterized, distinguished by congener selectivity and position o f the removed chlorine substituents, show reductive dehalogenation o f ort/zo-substituted chlorines. B y contrast, aerobic bacteria co-transform P C B s v i a catabolic pathways. Most o f the isolated aerobic PCB-degrading bacteria can only transform up to  tetra-  chlorobiphenyls, although some o f 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 o f the P C B content i n contaminated sites is thus resistant to aerobic microbial mechanism. A potential way to overcome this is the sequential treatment o f 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 o f 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, o f the aromatic substrates,  transforming the latter to one o f a limited number o f  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 o f 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 o f benzoic acid depends on the substituent present on its aromatic ring. From (92). P C B s are aerobically co-transformed v i a the bph pathway, which is responsible for the catabolism o f biphenyl (71, 81). The initial reaction i n the upper pathway is catalyzed by biphenyl dioxygenase ( B P D O ) , a multicomponent enzyme consisting o f 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 B p h F transfer electrons from N A D H to  B p h A E , which transforms biphenyl to cis-(2R,3S)-dihydrodihydroxy-l-phenylhexa-4,6diene. The latter is dehydrogenated and then oxygenolytically cleaved by biphenyl-2,3dihydrodiol dehydrogenase (BphB) and 2,3-dihydroxybiphenyl-l,2-dioxygenase (BphC), respectively. The ring cleavage product is then hydrolysed to benzoate and 2-hydroxy2,4-dienoic acid by the hydrolase B p h D (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; B p h B , biphenyl-2,3-dihydrodiol  dehydrogenase;  BphC,  2,3-  dioxygenase; B p h D , 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate  dihydroxybiphenyl-1,2hydrolase.  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 o f them can grow on mono- and dichlorinated biphenyls and most cometabolize more highly chlorinated biphenyls using biphenyl as a growth substrate. The number and position o f substituents i n the biphenyl ring affect the rate o f 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 o f aerobic bacteria are very restricted. Engineering this pathway to degrade a greater variety of P C B s is one o f the most promising approaches i n bioremediation.  Effective  engineering requires better knowledge o f 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 twocomponent 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 o f 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 o f a system to ensure strong induction under different environmental conditions.  1  owa  A>  M  4.1  M  a  c  K  ORfP  At  A2 OR? A3  M  B  C  D  aap  l^lllJilllill>^P»r^^  k b  <—!  £  C  F^^P? ^^ 1  ttlJ Oxygenase a-sutjunit Oxygenase p-subunit B § Fftiradoxin fSB Oxittoreductnse  ^  K  lUUlUUD^B^m SS52HZ& I  0««  /»»  A3  A3  B  C  O  Bij^enyWhydrocliol dehydrogenase E2^ 2,3-Oihydr£>xybiphenyl 1,2-cioxygenase d} HOPOA hydrolase (B) R«gulator  1  n  LB400  BNimiimitssssrM^  S  4  H  Oftfj  A4  F  7  1  5  • ' D P RHA1  — > H T r H m KKS102 ES GlutalWsfi* transferase 2-Hydroxypen!a.2,<*.<iienoate hydrata&e O Acotakiehyde dehydrogenase g3 4-Hydrt!xy-2-e^alerate aMolasa  Figure 1.4. The bph operon in different bacterial strains. The organization o f the bph genes i n the following bacteria: B. xenovorans L B 4 0 0 , 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 i n which both atoms o f dioxygen are incorporated into the product (18). Three general classes o f dioxygenases have been described: lipoxygenases catalyze the hydroperoxidation o f polyenes; ringcleaving dioxygenases catalyze the carbon-carbon bond fission o f catecholic compounds; and ring-hydroxylating dioxygenases catalyze the cz's-dihydroxylation o f arenes.  1.3.1 Ring-hydroxylating dioxygenases Ring-hydroxylating dioxygenases, o f 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 o f the oxygenase. Iron-sulfur clusters are common i n 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 o f sulfur and iron are ideal for accepting, donating, and storing electrons.  6  Iron-sulfur  clusters occur in one o f four configurations i n biology: as a  mononuclear Fe ligated to 4 cysteines, rhombic [2Fe-2S], cuboidal [3Fe-4S], and cubane [4Fe-4S] clusters (14). The ligands o f the [2Fe-2S] cluster are most commonly four cysteines.  However, i n the Rieske-type [2Fe-2S] clusters the ligands are two histidine  (His) residues and two cysteine (Cys) residues. Formally, each iron center i n 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 o f 2+ and 1+, respectively (57). In Rieske-type clusters, the redox active iron is coordinated to two H i s residues. Two groups o f Rieske FeS proteins have been recognized: those possessing high and low reduction potentials, respectively.  High-  potential Rieske proteins, typified by the cytochrome be \ complex o f the respiratory chain, have pH-dependent reduction potentials, attributed to coupled deprotonation o f the H i s ligands. O n the other hand, low-potential Rieske proteins, such as B p h F , have p H independent potentials (24). The non-heme iron centres are also involved in many different  biological  reactions. They employ a variety o f 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 o f 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 i n their structure, mechanism and cofactor requirements. They were first classified i n three classes, based on the number o f components and the nature o f 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 o f the cc-subunit o f the oxygenase, which reflects its phylogeny (68, 96). Both systems allow the classification o f any dioxygenase within groups possessing certain catalytic characteristics and specificity. Moreover, both classification systems have  identified four  preferentially  major  transform  subfamilies  toluene/biphenyl,  o f ring-hydroxylating dioxygenases naphthalene,  benzoate  and  that  phthalate,  respectively.  7  1.3.3 Proposed catalytic mechanism of ring-hydroxylating dioxygenases In  the proposed  catalytic mechanism  o f ring-hydroxylating dioxygenases  (reviewed i n (21), Figure 1.5), the mononuclear Fe center and the Rieske cluster o f the enzyme start i n the oxidized state. The activation o f dioxygen b y the dioxygenase requires binding o f the aromatic substrate close to the mononuclear Fe center and the reduction o f both metallocenters. The binding o f O2 to the reduced mononuclear iron generates a superoxide intermediate. This intermediate has been proposed to react i n one o f two ways. In the stepwise mechanism (A), the superoxide reacts with the aryl substrate to yield a bridged iron-alkyl peroxo species. Fission o f the 0 - 0 bond yields a monohydroxylated alkyl species and an iron ( V ) species which would effect the second dihydroxylation on the same face o f the alkyl substrate. In the concerted mechanism (B), the cleavage o f the 0 - 0 bond cleavage occurs first, yielding an 0 = F e ( V ) - O H intermediate that would effect dihydroxylation i n a single step.  Figure 1.5. The proposed stepwise (A) and concerted (B) mechanisms for ringhydroxylating dioxygenases (from (21)).  8  More recent studies have yielded the crystal structures o f ternary complexes o f naphthalene dioxygenase ( N D O ) 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 o f 1.8 and 2.0 A. The aryl substrate is positioned slightly further from the iron, with distances o f 3.3 A (C2) and 2.9 A (C3) from the bound oxygen. The structures o f N D O with its substrates support a concerted mode o f attack, i n which both oxygen atoms are polarized similarly and react with the carbon atoms o f the substrate double bond (Fig. 1.6). This reaction would explain the cz's-stereospecific addition o f both oxygen atoms to substrates by N D O and other dioxygenases.  y V V „ N o—T*C  Ferredoxin e -  u  [2Fe2S]  Air oxidation  ;  (1)  (2)  Substrate Ferredoxin //Product e-  N  O—Fs  -  (3)  OH.H  [2Fe2S]  (  (6) Substrate 2H  (4)  +  i  r  v •o  [2Fe2S]^  (5) 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 o f the latter is coupled to the consumption o f 0 . Uncoupling 2  occurs when the activated oxygen is unable to react with the aromatic substrate because o f 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 o f 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 i n cytochrome P450 m, i n which the transformation o f camphene, an analogue o f camphor, ca  is uncoupled from the oxygen consumption. Crystallographic studies indicate that camphene is highly mobile i n the active site, allowing the entry o f solvent. The latter acts as a source o f protons, leading to the production o f H2O2 and/or H2O from the activated 0  2  (74).  Figure 1.7. Proposed routes of uncoupling in ring-hydroxylating dioxygenases. The nature and number o f activated oxygen intermediate(s) are unknown. The formation o f O2", H2O2 and H2O consume 1, 2 and 4 reducing equivalents, respectively.  10  The investigation o f the relative degree o f uncoupling with different substrates provides a valuable insight i n the binding o f the different P C B substrates to the active site. Uncoupling in B P D O B 3 5 6 and B P D O B 4 O O by certain P C B congeners proceeds mostly L  via formation o f H2O2 (48, 60). This production o f H2O2 has also been observed i n N D O (56) using benzene as a substrate, suggesting a similar mode o f action i n both enzymes.  1.4 Biphenyl dioxygenase (BPDO) B P D O is the first enzyme i n the biphenyl degradation pathway and is a typical three-component,  ring-hydroxylating dioxygenase. A s summarized i n Figure 1.8, the  enzyme catalyzes the insertion o f molecular oxygen into the aromatic substrate nucleus forming cw-(2i?,35 )-dihydroxy-l-phenylcyclohexa-4,6-diene. Each a subunit o f 013P3 f  oxygenase contains a Rieske-type Fe S2 cluster and a mononuclear iron center, which 2  represents a total o f 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 i n B p h G , the Rieske iron sulfur cluster i n B p h F and the Rieske center i n the terminal oxygenase (50). The mechanism o f dihydroxylation is thought Pseudomonas  sp. N C I B  9816-4  to very similar to that o f N D O from  (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 o f a ring-hydroxylating oxygenase was that o f N D O from Pseudomonas strain N C I B 9816-4 (50). To date, the published crystal structures o f related oxygenases include those o f cumene dioxygenase from Pseudomonas fluorescens IP01 ( C D O ) , 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 o f the B P D O oxygenases from C. testosteroni B356 ( B p h A E  B 3 5 6  ) and B. xenovorans L B 4 0 0  (BphAELB4oo) have been solved, but not published (1, 48). B P D O s from B. xenovorans L B 4 0 0 and P. alcaligenes K F 7 0 7 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 o f 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  BPDO 356  BPDOLB400  BPDOKF707  BPDORHAI  CDOJS375  BPDOLB4OO  75  100  95  68  74  33  32  BPDO 356  100  76  76  66  76  33  33  B  B  ND0  9 8 1 6  4  NBD0 76S JS  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 o f the large and small subunits. Each sequence is identified according to the strain and dioxygenase to which the oxygenase belongs. The quaternary structure o f the  013P3  oxygenase resembles a mushroom (Fig. 1.9)  in which the a subunits, arranged i n 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 b y 43 A. However, the [2Fe-2S] cluster from one subunit interacts with the mononuclear iron center o f the neighbouring subunit, at a distance o f 14 A.  12  Figure 1.9. The structural fold of  BPDOB356-  The quaternary structure resembles a  mushroom i n 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 o f the metallocentres are completely conserved among oxygenases. In BphAE 356, the ligands o f the Rieske-type cluster are CyslOO, C y s l 2 0 , H i s l 0 2 and B  H i s 123, all o f which are located i n the N-terminal half o f the a subunit. The sulfur atoms i n 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 T r p l 2 5 , conserved among various B P D O s . In N D O , however, the residue corresponding to M e t l 0 5 is L y s . The ligands o f the mononuclear iron in BphAEB356 are His233, His239 and Asp388, all o f which are located i n the C-terminal half o f the a subunit. The A s p can apparently ligate the iron i n different ways: i n BphAERHAi it is monodentate (35) whereas in N a h A E (50) and BphAE 356 (1) it is bidentate. In N a h A E , a fourth residue, Asn201, B  13  occurs close to the mononuclear iron. The distance o f 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 o f 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 o f the iron, since all mutants were active (1). However, the substitution Gln226Glu led to a high degree o f uncoupling and substrate inhibition when biphenyl was used as a substrate. This suggests a role o f 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 o f the metallocentres i n the oxygenases are very similar and are spanned by a conserved Asp (230 in BphAE 356)- This suggests that the electron transfer between the [2Fe-2S] B  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 NDO  (49). In BphAERHAi,  a dioxygen molecule can be  accommodated  in the  corresponding space (35). Nevertheless, a series o f conformational changes occur upon substrate binding in BphAERHAi that were not observed in N D O . Interestingly, most o f these changes occur around just one o f 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 i n this binding pocket. It is possible that the binding o f 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 b y Asp388, His233 and His239, whereas one iron o f the Rieske cluster is coordinated b y C y s l 0 0 and C y s l 2 0 and the other is coordinated by His 102 and H i s 123. Purple and orange spheres represent the iron and sulfur atoms, respectively.  Further  details about other residues are described i n the text.  1.4.2. Substrate preference of BPDO A s the first enzyme i n the bph pathway, B P D O is a major determinant o f 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 i n (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) i n 1990. The authors attributed differences i n the degradation rates o f 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 o f the non-hydroxylated ring did not influence congener reactivity. Class III preferentially transformed para-  over  ortho-  15  substituted congeners; and class I V enzymes, represented by those o f B. xenovorans L B 4 0 0 and Ralstonia  eutropha H850, degrade a broad range o f congeners and exhibit  both 2,3- and 3,4-dihydroxylation activity. The reactivity preference o f class I V enzymes was ortho > meta > para chlorobiphenyls. It is now common to classify B P D O s as having either K F 7 0 7 - or LB400-type reactivity. These two B P D O s have been extensively studied and show very different reactivities (37). B P D O L B 4 0 0 transforms a wide range o f congeners, including some containing up to six chlorines (17, 65). A s noted above, B P D O L B 4 0 0 preferentially transforms ort/zo-substituted congeners and catalyzes the 3,4-dihydroxylation o f certain congeners  with chlorines at positions 2 and 5, such as 2,2',5,5'-tetraCl biphenyl.  BPDOLB4OO is also remarkable i n that it catalyzes the dehalogenation o f certain 2-C1 congeners (38, 80, 82, 83). The product o f this reaction is a 2,3-dihydroxybiphenyl ( D H B ) , obviating the need for B p h B . 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 O K F 7 0 7 transforms a narrow range o f substrates and catalyzes neither 3,4-dihydroxylation nor ortho-dechlorination. Moreover, it preferentially transforms either 3,3'- or 2 , 2 ' - d i C l biphenyl (37).  4 , 4 ' - d i C l biphenyl over  In addition to the fundamentally  reactivities o f BPDOLB4OO and B P D O K F 7 0 7 ,  different  the regiospecificity and efficiency o f  transformation depends i n a complex fashion on the substitution patterns o f both aryl rings (83). Indeed, it has been suggested that the enzyme's substrate preference depends more on the relative position o f 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 o f substrate preference i n B P D O s and have generated variant enzymes possessing enhanced PCB-transforming activities. Such studies have been facilitated b y the very similar amino acid sequences o f BPDOLB4OO and BPDOKF707  despite their different reactivities. Thus, their respective BphFs and B p h G s  have identical sequences whereas B p h A and B p h E share 95.6% (20 differences i n 460 residues) and 99.5%o sequence identity, respectively (29, 88). Mondello and Erickson compared the sequences o f BphA B400 and BphAicF707 and used directed mutagenesis to L  identify four regions (I, II, III, and I V ) whose sequences influence the range o f congeners attacked (66). A l l four regions occur i n 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 i n region III o f BphALB400 improved the ability o f the enzyme to  16  transform 4 , 4 ' - d i C l biphenyls, although the highest improvements i n activity were achieved by multiple substitutions in this region, suggesting a cooperative or additive effect. The importance o f these residues has since been confirmed by K i m u r a et al. and Barriault et al. (4, 6, 52). A l s o , resolution o f the crystal structure o f B p h A E revealed that residues i n region III are located in the upper part o f the substrate pocket. K i m u r a et al. also reported an expansion o f the degradation capability o f K F 7 0 7 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 o f substrate recognition are in  B p h A , there are also reports that B p h E , the small subunit o f the oxygenase, can  influence substrate preference to some extent (33, 42, 52). Suenaga  et al. developed a three-dimensional model o f  B P D O K F 7 0 7 based on the  crystal structure o f N D O and created a series o f site-directed mutants with changes i n amino acid residues located close to the catalytic non-heme iron center, including residues 335 and 341 (87). Replacement o f Ile335 by the corresponding Phe from L B 4 0 0 allowed the 3,4-dihydroxylation o f 2,2',5,5'-tetraCl biphenyl by the mutant enzyme. Interestingly, the Phe227Val and Phe377Ala mutants exhibited a novel transformation o f 3 , 3 ' - d i C l 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 o f B P D O L B 4 0 0 ,  proving that residues that are not in the immediate vicinity o f the substrate can significantly influence the structure o f the active site (98, 99). Directed evolution, which mimics the natural evolutionary processes o f genetic mutation, recombination and selection (25), has been applied to great effect i n B P D O . Shuffling o f  bphAiBm and bphA^joj yielded variants o f the enzyme with very different  activities and also supported the important role o f regions III and I V i n determining substrate preference (52). Gene shuffling between the L B 4 0 0 and the B356 oxygenases has also confirmed the important role o f the C-terminal portion o f the a subunit (5). Two of the variant enzymes characterized i n this thesis, BphAEng and BphAEmo, were previously created b y shuffling a targeted region o f  bphAmse and  6/?/JALB4OO (4).  BphAEn9 and B p h A E m o essentially have the same sequence as BphAEL 4oo except that B  region III is replaced with that o f BphA 356- The two variants differ by a single residue at B  position 267 ( A l a i n BphAE B400 and BphAEng, and Ser i n BphAE 356 and BphAEmo; L  B  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 o f them. M o r e recently, Barriault  et al. constructed a library o f B P D O L B 4 0 0 mutants i n which some o f the variants  17  exhibited a different regiospecificity against 2 , 2 ' - d i C l biphenyl, with 3,4-dihydroxylation preferred over 2,3-dihydroxylation and subsequent dechlorination (6). This study proved the influence o f the residues i n region III not only in the overall activity o f the enzyme but also on its regiospecificity toward different congeners.  Residue numbering 267  335  336  338  341  Ala  Thr  Phe  Asn  He  Ala  Gly  He  Thr  Thr  BphAmo  Ser  Gly  He  Thr  Thr  BphA 56  Ser  Gly  He  Thr  Thr  BphA B4oo L  BphA  n9  B3  Table 1.2. Comparison of BphA sequences at key positions in variants 119 and 1110. The table shows only those residues that were substituted i n BphAng and BphAmo with respect to BphA B400 (4). Thus, the sequence o f BphAmo and BphAng at these positions is L  identical to that o f BphA 356B  The fact that relatively small changes i n these highly conserved sequences o f the L B 4 0 0 and K F 7 0 7 enzymes result i n significant changes in substrate preference makes B P D O one .of the best targets for engineering bacteria with better PCB-degrading capacities. Engineering o f 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 o f B P D O K F 7 0 7 catalyzed the  angular  dioxygenation o f dibenzofuran, an uncommon dioxygenase activity (86). Changes i n the enzyme's  regiospecificity are  engineering  of  biphenyl  also o f great interest  dioxygenases  shows  a  for organic great  potential  synthesis. not  Thus,  only  for  bioremediation, but also for so-called "green chemistry".  18  1.5 Aim of this study G i v e n its importance in the bph pathway, B P D O is an attractive system to engineer.  The overall objective o f this study was to characterize four variants o f this  enzyme to gain a better understanding o f B P D O and to facilitate its engineering for improved P C B degradation. The variants include two w i l d type enzymes, BphAE 4oo LB  and BphAE 356, and two variants generated v i a directed evolution, BphAEng and B  BphAEino (4). The steady-state kinetic constants o f 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 o f uncoupling between O2 utilization and congener transformation was determined for the different enzymes using different congeners. P C B transformation products for some o f the most persistent congeners tested were characterized using mass spectrometry and N M R . Variant 119, B 3 5 6 and L B 4 0 0 were also used for crystallization studies, i n the absence and presence o f P C B congeners, to investigate the structural basis for changes i n enzyme function. This is the first study in which the activities o f anaerobically purified B P D O s , both engineered and w i l d type, are tested against against a range o f individual congeners as w e l l as against a mixture o f congeners.  The results are discussed i n terms o f the proposed catalytic mechanism o f  ring-hydroxylating dioxygenases and the crystal structure o f B P D O .  19  2. MATERIALS AND METHODS 2.1. Chemicals and reagents Biphenyl was purchased from Aldrich (Mississauga, O N ) . 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, O N ) . Restriction enzymes and T 4 D N A polymerase were purchased from N e w England Biolabs (Pickering, O N ) . Pwo D N A polymerase was purchased from Roche (Laval, Q C ) . Oligonucleotides were purchased from the N A P S Service unit at the University o f British Columbia (Vancouver, B C ) and I D T (Integrated D N A Technologies, Coralville, U S A ) . Acetonitrile, ethyl acetate and hexane (Fisher Scientific, Mississauga, O N ) were o f 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 o f bphAEng and bphAEmo, respectively, were generously provided b y D r . M i c h e l Sylvestre ( I N R S - I A F , Montreal, Q C ) . Strains harbouring pT7 derivatives were grown i n the presence o f ampicillin (100 ng/mi) or carbenicillin (15 ag/ml). Strains harbouring p P A I S C - 1 were grown i n 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 i n 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 o f the bphAE genes was then induced by the addition o f isopropyl-l-thio-PD-galactopyranoside (IPTG) to a final concentration o f 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. C e 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, Q C ) . 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 o f 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 o f 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 o f N  2  (< 2  ppm 0 ) using an M B r a u n Labmaster glovebox (Stratham, U S A ) . Chromatography was 2  performed on an A K T A Explorer 100 (Amersham Pharmacia Biotech, Baie d'Urfe, Q C ) interfaced to the glovebox to minimize the oxygen content o f the purification buffers and protein fractions (93). A l l buffers were prepared using water purified on a Barnstead N A N O p u r e U V apparatus to a resistivity o f greater than 17 MQ»cm. Buffers were sparged with N and equilibrated i n the glovebox for at least 24 h prior to use. 2  The washed cell pellet from 4 to 8 L o f culture was resuspended i n 40 mis o f 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, O N ) and disrupted by successive passages through a cell homogenizer (Emulsiflex C-5, Avestin, Ottawa, O N ) 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 i n 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  Reference  Relevant phenotype/genotype properties  Strains (39)  E. coli DH5ct  F",  E. coli C41[DE3]  Mutant of E. coli B L 2 1 [ D E 3 ] ;  E. coli L E 3 9 2 E. coli SGI3009  F,r,m  (pREP4)  F",  r", m  +  F",  (62)  r", m"  (24) (47)  r", m"  Plasmids (84)  pT7-7 and pT7-6  T7 promoter, C o l E l origin, A p  pT7-7AE3  pT7-7 carrying 6p/zAE 356  (48)  pT7-6a  pT7-6 carrying &P/ZAEFGBCLB400  (43)  pDB31  T7 promoter, p i 5 A origin, A p  (16)  pDB31-II9  pDB31 carrying bphAEuy  (4)  pDB31-II10  pDB31 carrying bphAEuio  (4)  PRKNMC  I n c P l replicon, lac promoter  r  B  r  (67, 89)  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 pPAISC-1  Pseudomonas aeruginosa P A 0 1 , T c  (67, 89)  r  pT7II9  pT7-7 carrying bphAE 9  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  This study  pEBRE12  p L E H P 2 0 carrying /3/?/ZFLB4OO  [8]  pQE31G  pQE31 carrying 6p/zG 356  [9]  U  bphAE 3 bphFGBCui4oo B  56  B  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'-CATGAAGCTTGTACCCCCTAGAAG AACTGC-3' 5'-P-TGTTTAACTTTAAGAAGGAGAT AT A C T C A T G A G T  B356-BspHI  TCGACTATGAAAGATACC-3'  22  The crude extract was divided into equal portions, each o f 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 o f 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 o f the Rieske-type [2Fe-2S] center, were concentrated to 5 m l b y ultrafiltration using a stirred cell equipped with a Y M 30 membrane (Amicon, Nepean, O N ) . 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 P S (buffer A containing 5% ammonium sulfate). The column was operated at a flow rate o f 1 ml/min. The oxygenases were eluted i n a decreasing ammonium sulfate gradient (5% to 0% over 4 column volumes). Brown-coloured fractions were concentrated to 10-15 mg/ml b y ultrafiltration, flash frozen as beads i n liquid N2, and stored at - 8 0 C .  2.4.2 Ferredoxin (BphF) His-tagged  ferredoxin  from  Burkholderia sp. L B 4 0 0  (ht-BphFLB40o) was  anaerobically prepared using the QIAexpress system from Qiagen (24). Briefly, the cell pellet from 4 L o f culture o f  E. coli L E 3 9 2 containing the plasmid p E B R E 1 2 was  resuspended i n 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 m i n and the cellular debris was removed b y ultracentrifugation at 45,000 g for 45 m i n 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-BphF B4oo was eluted with 20 m M L  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 - 8 0 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 o f culture o f E.  coli S G 1 3 0 0 9 ( p R E P 4 ) containing the p Q E 3 1 G plasmid was resuspended i n 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 m i n 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 , and stored at - 8 0 C . 2  2.5. Protein analysis S D S - P A G E was performed using a 12% resolving gel and a B i o R a d 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 o f a series o f dilutions o f 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-pphenylenediamine 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' )  Reference  1  BphAE 4oo  455  8.3  (38)  BphAE 356  455  10.1  (47)  BphAEn9  455  8.4  This study  BphAEmo  455  9.6  This study  BphF B400  326  9  (24)  BphG 356  450  11.8  (47)  LB  B  L  B  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 M o d e l 5301, Y e l l o w Springs, O H ) (93). The activity assay was performed i n a thermojacketted Cameron Instrument C o . model R C I respiration chamber (Port Aransas, U S A ) connected to a Lauda M o d e l R M 6 circulating bath. Data were recorded every 0 . 1 s and initial velocities were calculated from the slope o f the progress curve for each consecutive 6 s interval. The standard activity assay was performed i n a total volume o f 1.4 m l o f airsaturated 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 oo, 1.8 u M ht-BphG 56 and 0.6 u M B p h A E . The LB4  B3  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 i n the assay were  prepared  fresh  daily.  Stock solutions and protein samples  were  prepared  anaerobically. The electrode was zeroed on the day o f use by adding an excess o f sodium hydrosulfite to the buffer i n the reaction chamber. It was calibrated using standard concentrations  o f catechol and  an excess  o f catechol 2,3-dioxygenase.  Activity  determinations were corrected for the consumption o f O2 observed i n the absence o f oxygenase. One unit o f enzyme activity is defined as the amount o f enzyme required to consume 1 umol o f 02/min under the described conditions. Apparent steady-state kinetic parameters for biphenyl were determined by measuring rates o f oxygen uptake in the presence o f 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 o f L E O N O R A (23).  2.7. HPLC and GC-MS analyses HPLC  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, O N ) . The instrument was operated at a flow rate o f 1 ml/min. Biphenyls were eluted with a 20 m l gradient o f 50% to 90% acetonitrile i n H2O. Samples o f 100 ul were injected and the amount o f biphenyl was determined from the area o f absorbance peak at the appropriate wavelength using a standard curve. Standard curves for each biphenyl were established b y determining the  25  peak areas o f known amounts o f the biphenyl. A l l standard curves had correlation factors higher than 0.97. A l l standard samples were treated i n the same way as reaction mixtures to account for losses o f 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 m m X 30 m X 0.25 um) (Agilent, Mississauga, O N ) . The instrument was run at a flow rate o f 53.5 ml/min and a pressure o f 10.7 psi. Standard curves for each biphenyl were established by determining the peak areas o f known amounts o f 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 o f biphenyl or congener, 350 u M N A D H and the same concentrations o f B P D O components used i n 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 o f hydrogen peroxide  was estimated using catalase, 650 U o f which was added to the reaction mixture at the time corresponding to the acetonitrile quench. The amount o f oxygen detected by the oxygraph upon addition o f catalase was taken to represent 50% o f the total hydrogen peroxide produced during biphenyl transformation.  The consumption o f biphenyl was  determined b y H P L C (section 2.7).  2.9. Depletion of PCB mixtures by purified BPDOs Depletion assays were performed i n 12 m l glass vials sealed with teflon caps in a total volume o f 1.0 m l o f air-saturated 50 m M M E S , p H 6.0 (25°C). The reaction mixture contained the same concentrations o f B P D O components as the standard oxygraph assay, 350 u M N A D H , and 10 u M each o f 3,3'-diCl, 4 , 4 ' - d i C l , 2,6-diCl, 2,3,4'-triCl, 2,3',4triCl, 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% o f 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 b y G C - M S .  26  2.10. Whole-cell assays E. coli C41(DE3) cells freshly transformed with the isc plasmid and either pT76a, 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 2 2 ° C to an OD600 o f 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 i n the same buffer at an OD600 o f 2.0. One-ml portions o f this suspension were distributed i n 12-ml glass vials with teflon caps. Each vial received 10 u,M o f each congener i n 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 i n 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 o f 10 u M . The samples were extracted twice with 1 m l o f hexane, pooled, dried over sodium sulphate and transferred to G C vials. The P C B content was analyzed as described i n section 2.7. Protein levels i n the different strains were verified using Sypro Ruby-stained denaturing gels ( S D S polyacrylamide) o f whole cells. Band intensities were quantified with ImageQuant 5.2 (Amersham Pharmacia).  The levels o f each o f 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 BPDO 356 B  To  characterize the  BPDOe356-catalyzed transformation product  o f 2,4,4'-  trichlorobiphenyl, two reactions were carried out i n parallel. Each contained 600 u M o f 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 o f nitrogen. The sample was resuspended in acetone-^6 and analyzed using a 500 M H z Varian N M R spectrometer (Department o f Chemistry, UBC).  27  3. RESULTS 3.1. Purification and characterization of BPDO Relevant details o f the anaerobic purification o f the oxygenase components o f B P D O are summarized i n Table 3 . 1 . The enzymes were estimated to be greater than 9 0 %  pure as judged b y S D S 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 B p h A E L ^ o o and BphAE 356 had specific activities o f 0 . 2 B  and 4 U/mg, respectively. BphAEng and BphAEmo had specific activities o f 0 . 6 and 0 . 3 U/mg,  respectively. T o ensure the optimum activity o f 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, BphAE B 356,  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 , BphF 400 ( 1 2 k D a , 1 5 |ag). LB  Lane 7 , B p h G  B35  6 ( 4 3 k D a , 1 5 ug).  28  Table 3.1. Purification of the oxygenases  Enzyme/Purification step  Total protein  Total activity  Specific activity  Yield  mg  U  U/mg  %  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  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  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  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  BphAE 4uo LB  Crude extract  BphAE B356 Crude extract  BphAEno Crude extract  BphAEmo Crude extract  Activity units (U) are described i n Materials and Methods. Standard deviations (n = 4) are indicated i n parentheses.  29  The sulphur content o f 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 o f an oxidized Rieske-type center [2Fe-2S] cluster, with maxima at 323 and 455 nm and a shoulder at 575 n m (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" cm" , respectively (based on 1  1  sulphur content).  <  0-1  310  ,  ,  ,  r-  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 o f 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 o f the initial rate o f oxygen uptake on the concentration o f biphenyl. To estimate the apparent steady-state kinetics parameters, the concentration o f 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 i n Figure 3.3.  30  BPDO  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 o f the data to the Michaelis-Menten equation using the least squares, dynamic weighting options o f L E O N O R A . Similar results were obtained using different enzyme preparations. The fitted parameters for experiments were K = 20 ± 4 u M and V m  BPDOB356  averaged over 5  = 150 ± 10 p-M-min" . The averaged fitted 1  m a x  parameters for BPDO119 (n = 5) were K = 0.3 ± 0.2 u M and V m  = 24 ± 2 n M m i n " . A l l 1  m a x  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 i n Table 3.2. O f the four variants, the specificity constant (k /K ) o f BPDOLFMOO for biphenyl was cai  m  highest, and was approximately 10-fold greater than that o f B P D 0 B 5 6 - Interestingly, 3  BPDOB356  exhibited the highest turnover number, k , but its K  m  cat  was a hundred times  higher than that o f B P D O L B 4 0 0 - W i t h respect to these parameters, BPDO119 and B P D O m o fall between the two natural variants. However, BPDO119 is more similar to BPDOLB4OO while B P D O m o is more similar to B P D O B 3 5 6 In each o f the four enzymes, the consumption o f O2 was well coupled to the consumption  o f biphenyl (Table 3.2).  That is, in the  presence o f a  saturating  concentration o f biphenyl (150 u M ) , the amount o f biphenyl consumed corresponded to the amount o f 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 BPDO 356? BPDO119 and BPDOmo using biphenyl as substrate. B  BPDOLB400>  k /K (\10 M'Y ) Biphenyl:02  BPDO  ^m(uM)  B356  20 (4)  4.1 (0.2)  0.21 (0.04)  1.0 (0.2)  ND  LB400  0.18(0.03)  0.4 (0.1)  2.4 (0.7)  1.1 (0.1)  ND  119  0.3 (0.2)  0.67 (0.08)  2(1)  1.0 (0.1)  ND  mo  2(1)  1.03 (0.05)  0.5 (0.3)  0.9 (0.2)  ND  6  ca  1  m  H 0 :0 2  2  2  Experiments were performed using 50 m M M E S p H 6, 25°C. The values o f 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 i n "Materials and Methods". N D , not detected.  32  3.3. The reactivity of BPDO variants with individual chlorinated biphenyls The reactivity o f each B P D O with each o f five different chlorinated biphenyls was examined over two minutes at a single congener concentration (50 u M ) . Biphenyl depletion was followed by H P L C and the O2 depletion was followed with the oxygraph. The percent o f depletion o f the different substrates by each o f the four enzymes is summarized i n Table 3.3. O f the four isozymes, B P D O 3 5 6 showed the best congenerB  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 3 5 6 depleted 2 , 2 ' - d i C l biphenyl just as well as B  BPDOLB400-  B y contrast, B P D O m o had the poorest ability to transform  congeners,  significantly depleting only 2,3'- and 3,3-diCl biphenyls. The overall congener-depletion activity o f BPDO119 was intermediate between that o f the two parental enzymes, except for 4 , 4 ' - d i C l biphenyl, which BPDO119 transformed more slowly than either parental enzymes.  Table 3.3. The depletion of individual chlorinated biphenyls by purified BPDOs % Depletion Congener  BPDO 56  BPDO 400  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  B3  LB  Assays were performed using 50 m M M E S , p H 6, 25°C. Each substrate was tested individually using an initial concentration o f 50 u M . The depletion o f the substrates was followed b y H P L C . Reactions were stopped after 2 min. A l l assays were performed i n 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 o f the four enzymes transformed any o f the tested congeners significantly faster than biphenyl. Except for biphenyl and, i n the case  33  of BPDO119 and BPDOLB400, 2 , 2 ' - d i C l biphenyl, the consumption o f the congener and O2 was uncoupled. In most cases, the O2 that did not react with the corresponding P C B was detected as H C>2 i n the reaction. However, not all uncoupling resulted in H2O2 2  production, suggesting that there is also some H2O production. Considering both the activities o f the enzymes towards the different congeners and the uncoupling values, the apparent substrate preference o f each enzyme at 25°C is as follows: B P D C W biphenyl > 3,3'-diCl > 2,4,4'-triCl > 4 , 4 ' - d i C l ~ 2 , 2 ' - d i C l > 2,6-diCl BPDOUMOO: biphenyl ~ 2 , 2 ' - d i C l > 3,3'-diCl > 4 , 4 ' - d i C l > 2,4,4'-triCl ~ 2,6-diCl BPDO119: biphenyl > 2 , 2 ' - d i C l > 3,3'-diCl ~ 2,4,4'-triCl > 2,6-diCl > 4 , 4 ' - d i C l B P D O m o : biphenyl > 2 , 2 ' - d i C l > 3,3'-diCl  34  Table 3.4. The reactivities of purified BPDOs with individual congeners BPDO356  BPDO 40o  B  BPDO„  LB  BPDO,„o  9  Act (nmol bph/min)  Bph:0  ND  12(4)  1.1(0.1)  ND  44(2)  0.7 (0.2)  0.2(0.04)  6(2)  0.4 (0.3)  0.3 (0.1)  6(2)  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)  2,2'-diCl  15(1)  0.33 (0.03)  0.6(0.2)  15(4)  1.0(0.3)  ND  12(2)  0.6 (0.2)  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)  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)  Congener  Act (nmol bph/min)  Bph:0  Biphenyl  55.3 (0.6)  1.0 (0.2)  3,3*-diCl  32.2 (0.2)  2,4,4'- triCl  2  H 0 :0 2  2  2  2  H 0 :0 2  2  2  Act (nmol bph/min)  Bph:0  2  1.0 (0.1)  Act (nmol bph/min)  Bph:O  25(1)  0.9 (0.2)  ND  10(1)  0.8 (0.03)  0.6 (0.3)  0.5 (0.1)  ND  ND  ND  ND  16(4)  1.2 (0.4)  0.4 (0.1)  ND  ND  ND  ND  ND  ND  H 0 :0 2  2  2  ND  0.26 (0.05) 0.31 (0.01)  0.05 (0.01) 0.15(0.05) 0.6 (0.2)  0.4 (0.2)  z  H 0 :0 2  Assays were performed using 50 m M M E S p H 6, 25°C. Each substrate was tested individually, with a final concentration o f 50 u M . The depletion o f 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 i n parentheses. The amount o f H2O2 detected upon addition o f catalase was taken to be 50% o f the total H2O2 produced during the reaction.  2  2  3.4. Characterization of the transformation product of 2,4,4'-triCl biphenyl by BPDO 356. B  A s shown in Table 3.3, B P D O B 3 5 6 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 o f 2,4,4'-triCl biphenyl by B P D O B 3 5 6 was purified by H P L C and absorbed maximally at  X = 293.5 nm, which is within the range o f X  max  of  other dihydrodihydroxydiols (3). Moreover, the incubation o f 2,4,4'-triCl biphenyl with the first three purified enzymes o f 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  BphAEFGC  (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 o f B p h A E was analyzed by N M R (Figure 3.4). The chemical shifts and coupling constants o f the protons were consistent with the presence o f four protons on the non-aromatic ring (excluding the hydroxyl protons), which are upfield o f the aromatic ring due to the shielding effect o f the - O H groups. The values are comparable to theoretical ChemDraw Ultra 7.0 (CambridgeSoft, obtained  for  transformation  similarly  chlorinated  values predicted  using  Cambridge, U S A ) as well as to the values 2,3-dihydroxylated  product was thus identified as  compounds  (7,  75).  The  2,3-dihydro-2,3-dihydroxy-2',4,4'-triCl  biphenyl (Figure 3.4), indicating that B P D O B 3 5 6 catalyzes the 2,3-dihydroxylation o f the congener on the monochlorinated ring.  36  ci  Figure 3.4. The NMR spectrum of the product of 2,4,4'-trichlorobiphenyI dihydroxylation by BPDO 56. *H N M R (300 M H z , acetone-d , 8): 4.5 (1H, d, J= 6 H z , B3  6  Hr/ H >), 4.95 (1H, d, J= 6 H z , H >/H >), 6.12 (1H, d, J= 6.1 H z , H V H -), 6.43 (1H, d, J= 3  2  5  3  6  6.1 H z , H V H - ) , 7.65 (2H, H / H / H ) , 7.5 (1H, H / H / H ) . The data collected were not 5  6  3  5  6  3  5  6  enough to assign protons 3, 5, 6, 5' and 6' to a specific chemical shift. The intensity o f the peak for H 7 H - was affected by the water signal suppression specified in the 2  3  instrument.  3.5. The depletion of a PCB mixture by purified BPDOs The activities o f the purified B P D O s were investigated using a mixture o f 8 congeners described by Barriault et al. (4). This mixture contained 10 u M each o f 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 o f each congener in the mixture after 20 m i n is summarized i n Table 3.5. Consistent with the assays performed with individual congeners,  BPD0B 56 3  showed the best congener-transforming activity: it was the only variant that significantly depleted all congeners and, with the exception o f 2,3,4'-triCl and 2,2',5,5'-tetraCl biphenyls, transformed each congener faster than any o f the other variants. Indeed, B P D O 5 6 was the only variant that detectably depleted 2,6-diCl, 3,3'-diCl and 4,4'-diCl B3  biphenyls. B y contrast,  BPDOLBMO  detectably depleted only 4 o f the congeners in the  mixture. However, it depleted 2,3,4'-triCl and 2,2',5,5'-tetraCl biphenyls faster than BPDO  B35  6 - The activity o f B P D O  n 9  was similar to that o f BPDO 4oo while BPDOmo had LB  37  the lowest overall depletion activity. Nevertheless, both BPDO119 and B P D O m o 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  BPDO 356  BPDO 400  BPDO  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)  B  LB  u9  BPDOmo  Assays were performed using M E S p H 6, 25°C. The mixture contained the 8 listed congeners.  The depletion o f the biphenyl substrates was followed b y 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 o f BPDOLB400, BPDO119 and B P D O m o 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 i n the enzyme assays except that the levels o f depletion were higher i n the former (Table 3.5). Thus, the substrate preference o f B P D O L 4 O O was 2,3',4-triCl > B  2,2',5,5'-tetraCl ~ 2,6-diCl.  2,2',3,3'-tetraCl > 3 , 3 - d i C l ~ 2,3,4'-triCl > 2,4,4'-triCl > 4,4' d i C l >  A s i n the purified enzyme assays, BPDO119 depleted most o f the tested  congeners faster than BPDOL 4OO> and displayed a degradation pattern more similar to B  that o f B P D O B 3 5 6 in purified enzymes assays. Whole cells containing B P D O m o did not degrade any o f the congeners  38  Table 3.6. The depletion of a PCB mixture by E. coli cells expressing BPDOs.  % Depletion BPDO 4oo LB  Congener  BPDOmo  BPDOIW  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 (£»/?/zAE B4oo, bphAEug or bphAEmo) and L  /3P/JFGBCLB400- Cells were grown to an OD600 = 1.0, harvested and resuspended to an  OD600 o f 2.0 i n 50 m M sodium phosphate, p H 7.5 supplemented with 1 g/L glucose. The depletion o f the biphenyl substrates (10 u M each i n 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 B P D O m o were the same at 3 and 6 hours.  39  4. DISCUSSION In the current study, highly active preparations o f B P D O oxygenases were produced to better investigate the structural basis o f their respective reactivities. Accordingly,  BphAE 4oo, LB  heterologously expressed  in  BphAEn9,  BphAEmo  and  BphAE 356 B  were  each  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 o f the oxygenase (45). Yields o f B p h A E ranged from 5 to 10 mg o f highly active protein per litre o f cell culture (Table 3.1). The sulfur and iron contents o f the preparations indicated that each contained full complements o f their Rieske-type clusters and mononuclear Fe centers. Moreover, the absorbance spectra o f the oxygenases were characteristic o f a Rieske-type cluster (Fig. 3.2). The yields o f BphAEB 56 and 3  BphAEL 4oo and their specific activities in reconstituted systems correspond to those B  reported b y 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 o f iron and partial destruction o f the Rieske center, the specific activities o f the four anaerobically purified enzymes observed in this study were greater than those reported for aerobic preparations (0.09 U / m g and 2.4 U / m g for BphAE B4oo and BphAEB356, L  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 o f N A D H or production o f H O P D A in coupled assays with B p h B and B p h C . For well-coupled systems, the use o f an O2 electrode provides a reliable, continuous assay to follow the dihydroxylation o f biphenyl directly. For substituted biphenyls, the use o f an O2 electrode enables a measure o f the degree o f uncoupling between O2 and biphenyl consumption. In each o f the four studied BPDO  variants, 0  2  consumption was well  coupled to biphenyl dihydroxylation,  validating the conclusion that B P D 0 B 3 5 6 is the most active enzyme using biphenyl as a substrate, followed by B P D O , B P D O m o , and B P D O B 4 O O (Table 3.1). The higher n 9  L  specific activity o f B P D O B 3 5 6 over BPDOLB4OO has consistently been reported. However, preliminary results obtained with purified his-tagged BPDO119 and B P D O m o indicated a higher activity o f both mutants over B P D 0 B 5 6 (4). The current results are more reliable 3  as they were obtained using anaerobically purified preparations o f native (i.e., nontagged) proteins and a continuous assay.  40  Steady-state kinetic studies revealed that  BPDOLB400  has the highest apparent  specificity constant for biphenyl, followed by BPDO119, B P D O m o and  BPDOB356  (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  BPDOLB400  and  BPDOB356  had similar Km values for biphenyl (46). This  could reflect the influence o f the his-tag or the lower sensitivity o f the assay used. Interestingly,  BPDOB356  is the only variant whose  Vmax  was observed at concentrations o f  biphenyl that exceed the latter's solubility limit (45 u M ) . 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 o f the substrate from the solid phase to the active site. The kinetic parameters o f mutant BPDO119 and B P D O m o for biphenyl are within the range o f those o f 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 o f the parental enzymes for their natural substrate. Interestingly, the identity o f the residue at position 267 appears to have a much greater effect on the steady-state parameters for biphenyl than the identity o f the residues at positions 335, 336, 338 and 341 combined (Table 3.2). The abilities o f 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 o f 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 o f depletion o f the individual congeners was lower i n the presence o f 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 o f the purified B P D O components versus those i n the cell as the former where prepared anaerobically and were highly active. differences may be due to factors such as different relative levels o f the  The BPDO  components and different uptake o f the congeners by the cells. M o r e particularly, the ratio o f the expression o f the ferredoxin, reductase and oxygenase i n 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 i n the whole cell assay (0.53 u M ) and i n the i n vitro assay (0.6 u M ) . However, the local concentration o f 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 i n the whole cell assay, but it was three times higher i n the i n vitro assay. In  41  general, it is difficult to compare results obtained by different researchers and to establish the substrate preference o f B P D O s due to differences i n 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 o f the enzyme. The relative preference o f  BPDOLB400  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 i C l > 2,3',4-triCl > 2,2',5,5'-tetraCl ~ 2,2',3,3'-tetraCl > 2,3,4'-triCl ~ 3,3'-diCl > 4 , 4 ' - d i C l > 2,4,4'-triCl ~ 2,6-diCl biphenyl. More specifically, researchers  other  have reported that the enzyme dihydroxylates biphenyl and 2,2'-diCl  biphenyl at similar rates (32, 87). Moreover, the apparent preference o f purified BPDOLB400  for 2,2'- > 3,3'- > 4 , 4 ' - d i C l biphenyls agrees with previous reports using: (1)  whole-cell assays with either individual congeners or a m i x o f the three (8); (2) wholecell 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) i n a process that does not involve dechlorination, as previously reported (6).  In the current study, the  depletion o f 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 o f  BPDOLB4OO  to catalyze the 3,4-dihydroxylation o f  2,2',5,5'-tetraCl biphenyl has been widely reported. In whole-cell assays,  B. xenovorans  L B 4 0 0 depleted 2,2',5,5'-tetraCl and 2,2',3,3'-tetraCl biphenyls at similar rates (17, 37).  E. coli cells expressing  B y contrast,  BPDOLB400  preferentially depleted 2,2',5,5'-tetraCl  biphenyl (6). The transformation o f 2,4,4'- triCl and 2,6-diCl biphenyls by purified  BPDOLB400  has not been reported to date. Results obtained using whole-cell assays expressing BPDOLB400  and a mixture o f congeners are ambiguous regarding the activity o f the  enzyme toward double ;?ara-substituted congeners, such as 4 , 4 ' - d i C l and 2,4,4'-triCl biphenyl. Some studies report a total lack o f activity against such congeners (4, 6), others report a favoured attack o f 2,4,4'-triCl over 4 , 4 ' - d i C l biphenyl when both are present i n 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 BPDO B4oo, in agreement with the enzyme's reported L  preference for double or^o-substituted congeners (20). The depletion o f both 2,4,4'-triCl and 4 , 4 ' - d i C l biphenyls i n presence o f a mixture o f congeners was comparable i n this study, although depletion o f the former by whole cells was slightly higher. Results obtained with whole cells o f  B. xenovorans L B 4 0 0 in virtually identical conditions  (Barriault et al., personal communication) also showed a higher depletion o f 2,4,4'-triCl biphenyl, contributing to the ambiguity. Other than this, the results obtained with whole cells o f  B. xenovorans L B 4 0 0 verified the degradation pattern observed i n the current  study. Overall, the current results substantiate introduction o f  and extend the conclusion that the  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 B P D O L B 4 0 0 B y contrast, the substrate preference o f B P D O B 3 5 6 differed from that o f 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 i C l ~ 2 , 2 ' - d i C l > 2,6-diCl > 2,2',3,3'-tetraCl ~ 2,2',5,5'-tetraCl. Moreover, in assays using purified components, B P D O B 3 5 6 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 o f purified BPDO 356 for 3,3'- > 4,4'- > 2 , 2 ' - d i C l biphenyls (48). B  However, previous assays performed using whole-cell assays expressing His-tagged enzyme reported low activity o f the enzyme against most congeners i n 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 o f BPDO 356 against the 2,6-diCl and 2,4,4'-triCl B  biphenyls (Table 3.5) is o f particular interest and has not been reported prior to the current study. The metabolite produced by the hydroxylation o f 2,4,4'-triCl biphenyl by B P D 0 B 3 5 6 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 o f the enzyme (Barriault et al., personal communication). Studies with His-tagged preparations further confirmed that the activity o f B P D O B 5 6 was much higher than that o f either B P D O L B 4 0 0 3  or BPDOp4, a variant o f BPDOLB4OO that degrades most congeners at a higher rate than BPDOLB400 (6). Although the sequence BPDOLB4OO  identity between BPDO 356 and each o f B  and B P D O K F 7 0 7 is very high (75 and 76%, respectively), the reactivity o f  B P D O 3 5 6 appears to be closer to that o f B P D O K F 7 0 7 . B P D O K F 7 0 7 also shows a high level B  43  of depletion for both 2,4,4'-triCl and 4 , 4 ' - d i C l 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 o f 2,6-diCl biphenyl (personal communication). Each o f  BPDOB356,  BPDOLB400, BPDO119 and BPDOp4 catalyzed the formation o f two hydroxylation products, both o f which were dichlorinated. This implies that one o f these products is 3,4dihydroxylated. Quantification revealed that  BPDOB356  produced higher levels o f the two  products than any o f the other variants. This is the first report o f 3,4-dihydroxylation by BPDOB356-  B P D O 5 6 has not been as w e l l studied as B P D O L B 4 0 0 or B P D O K F 7 0 7 B 3  Moreover, most the previous studies have been conducted using whole cells, usually expressing His-tagged enzyme and using different mixtures o f congeners (5, 8, 44, 45). Clearly, these approaches have limited what we have learned about this enzyme. A particularly striking characteristic o f BPDO 356 revealed by this study is the B  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 o f 1:4, resulting from the 2,3dihydroxylation o f the respective rings (83). In principle, B P D O B 3 5 6 could also catalyze the hydroxylation o f both rings.  However, the preference o f the enzyme for meta-  substituted congeners may favour an attack i n the least chlorinated ring. A s for 2,3,4'triCl biphenyl, which is also degraded to almost twice the level o f any other congener, attack probably proceeds v i a 2,3-dihydroxylation o f the least chlorinated ring. The 2,4substitution pattern o f 2,3',4-triCl biphenyl is also present i n 2,4,4'-triCl biphenyl, which the enzyme 2,3-dihydroxylated at a high rate i n single congener assays (Fig. 3.4). The preference o f  BPDOB356  for 2,3',4-triCl biphenyl over 2,4,4'-triCl biphenyl is consistent  with its preference o f 3,3'-diCl over 4 , 4 ' - d i C l biphenyl. That is, congeners containing a meta-substituent on the attacked ring appear to better substrates, presumably due to a better fit i n the active site. Another interesting characteristic o f  BPDOB356  is that it transforms both 2,2',5,5'-  and 2,2',3,3'-tetraCl biphenyls at a rate similar to that o f most d i C l biphenyls tested (Table 3.5). Neither B P D O 5 6 nor B 3  BPDOKF707  have been reported to transform either  2,2',5,5'- or 2,2',3,3'-tetraCl biphenyls, even i n assays using purified enzymes (5, 28). In recent whole cell assays performed under essentially identical conditions to those o f the current study, Barriault  et al. confirmed the high activity o f  BPDOB356  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 o f BPDO119 was closer to that o f B P D O L B W O than that o f BPDO 356 (Tables 3.3, 3.5 and 3.6): biphenyl > 2 , 2 ' - d i C l > 2,3,4'-triCl > 2,3'4B  triCl > 2,2',5,5'-tetraCl > 2,2',3,3'-tetraCl > 3,3'-diCl ~ 2,4,4'-triCl > 2,6-diCl > 4,4'd i C l . This is consistent with the steady-state parameters for biphenyl, which were also closer to those o f 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 o f 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 i n that BPDOng-containing cells depleted most congeners faster than BPDOLB400-containing cells. The apparent substrate preference o f the enzyme in whole cell assays was thus similar to that observed previously (4). B P D O m o was the least active enzyme i n this study. Nevertheless, i n the mixed congener assay, the overall pattern o f congener degradation by the purified enzyme was similar to that o f BPDO 4oo and BPDO119 (Table 3.5). Interestingly, B P D O m o showed a LB  slightly improved ability to degrade BPDOLB400  3,3'-diCl biphenyl when compared to both  and BPDO119 (Table 3.4). This could indicate an important role o f Ala267 i n  the binding o f meta-substituted congeners. O n the other hand, the improved degradation of 2,4,4'-triCl and 2,6-diCl biphenyls by BPDO119 when compared to BPDO 40o was not LB  observed i n B P D O m o , suggesting again a crucial role o f Ala267 i n substrate binding. Finally, it is not clear w h y whole cells expressing B P D O m o did not degrade any o f the congeners, although this is consistent with previously observations (4). The coupling between the consumption o f O2 and the aromatic substrate reflects the ability o f 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 o f this reaction depends on the proximity between the two reactants i n the active site and the exclusion o f solvent molecules from this environment. B y analogy to what has been observed i n 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 o f solvent species with the activated oxygen species results i n uncoupling as depicted in Figure 1.7. In this study, the transformation o f P C B s was uncoupled from the consumption o f O2 in all but one case: the transformation o f 2 , 2 ' - d i C l biphenyl by B P D O L B 4 0 0 (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 w i l d type enzymes and BPDO119, the degree o f uncoupling was inversely related to the rate o f depletion, indicating a better fit o f these congeners into the active site. Results obtained with both B P D O L B 4 0 0 and B P D O B 3 5 6 and 2,2'-,3,3'-, and 4 , 4 ' - d i C l biphenyl are consistent with previous results obtained i n identical conditions (1, 60). In the case o f BPDO119, the rates o f depletion for 2,6-diCl, 3 , 3 ' - d i C l and 2,4,4'-triCl were comparable (within experimental error). The lower degree o f uncoupling in the presence o f 3,3'-diCl biphenyl indicates a better fit o f the congener into the active site. Conversely, the high level o f uncoupling observed i n the presence o f 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, i n which the rate o f depletion is under 2 nmol/min but the ratio F ^ C ^ C h is close to one. Similarly, the lower uncoupling observed i n the presence o f these congeners for BPDO119 indicates that they fit the active site o f 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 o f the former was twice that o f 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 o f B P D O m o did not allow for a thorough investigation o f the uncoupling. While it is difficult to assign specific roles to individual residues i n determining congener preference, consideration o f the crystal structures o f BphAEB356, BphAE B400 L  and BphAEiig (Kumar and B o l i n , personal communication), each free and i n 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 o f biphenyl, respectively (Fig. 4.1.). In BphAE B400, the residues that line the proximal portion o f the L  pocket are Gln226, Phe227, Asp230, Met231, His323, Pro334, Asn337, Asn338, Ser379 and Glu385 (Fig. 4.1.). Residues lining the distal portion o f 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 o f particular interest as they shift upon the binding o f biphenyl to BphAELB400 (Fig. 4.2.). The equivalent residues i n 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 o f BPDO 356, B  BPDOLB4OO, B P D O K F 7 0 7 ,  BPDORHAI,  and  CDOJS375 (data not shown). Moreover, residues 283 and 336 are Leu/Ile and He i n all o f these dioxygenases except BphAELB400- Residue 320 is not conserved at a l l , and residue 286 is conserved i n all but BphAEB356- T w o other residues that shift significantly upon the binding of biphenyl i n BphAELB400 are Val287 and Phe387.  Figure 4.1. The substrate-binding pocket of BphAE B4oo- Ligands to the iron are L  shown i n grey. Other residue i n the pocket is shown i n 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  BPDOLB400-  B y contrast, this residue is Thr i n 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 i n the first sphere o f the substratebinding pocket are shown as open red boxes (residues 226, 227, 231, 243, 277, 283, 287, 323, and 336). Regions I, II, III, and I V defined by Mondello et al. (66) are labeled with green Roman numerals. The residues that were substituted i n BphAEng and B p h A E m o occur i n two distinct parts o f the enzyme: residues 335, 336, 338 and 341 ( L B 4 0 0 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 i n sequence to region II identified b y 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 P C B s . 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 i n the active site. Replacement o f A l a by Ser may create a new arrangement i n that region by changing the interactions  among  residues. The different activities of B P D O m o and B P D O 3 5 6 , two enzymes that both have B  Ser267, highlight the importance o f sequence context on the influence o f a specific residue.  Figure 4.4. The substituted residues in BphA and BphAEmo. The residues that are II9  substituted i n B p h A i ^ with respect to BphALB4oo are shown i n blue (T335G, F336I, N338T, I341T) and the residue substituted i n BphAEmo with respect to BphAELB400 is shown i n red (A267S). The figure depicts the alpha subunit only. The C-terminal domain is brown, and the N-terminal, Rieske domain, at the back o f the molecule, is blue. Bound biphenyl is shown i n 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 o f the roof o f the substrate-binding pocket and are also part o f region III identified by Mondello, as discussed above. T h e sequence o f region III, T F N N T R I , is shared by BphAE B400 a n d BphAEHsso, from A. L  eutrophus H850 (66), two o f the most versatile enzymes described i n the literature. The c o r r e s p o n d i n g 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 i n BphAE]CF707  and C D O .  In  the  structure  o f the  BphAE B4oo:biphenyl complex, Phe336 is 4.3 A from C 4 o f the distal ring o f the bound L  biphenyl, while residues 338 and 341 are - 1 0 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 o f Phe336 to the bound b i p h e n y 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 C 4 o f distal biphenyl ring, replacement o f 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 o f Thr335. This observation is supported in this study by the higher levels o f transformation of 4 , 4 ' - d i C l and 2,4,4'-triCl by B P D O n when compared to B P D O 4 0 0 and by the lower 9  L B  level o f uncoupling with both congeners for BPDO119. The effect o f Phe336 on substrate preference has been widely reported and suggests a close interaction o f these residues with the substrate (20, 66). Replacement o f Ile335 i n BphAEicF707 by the corresponding Phe336 from BphAE B4oo did not only prevent the enzyme from hydroxylating 4 , 4 ' - d i C l L  biphenyl, but also induced the 3,4-dihydroxylation o f congeners with a 2,5-substituted ring, a characteristic o f B P D O B 4 0 0 (87). Barriault et al. recently reported similar effects L  on regiospecifity toward 2 , 2 ' - d i C l biphenyl (6, 7). Replacement o f Thr335 and Phe336 by A l a and Met/lie resulted in an increase in 3,4-dihydroxylation, which was also affected i n part by changes in Ile341. The crystal structure o f B P D O L B 4 O O suggests a more subtle effect o f mutations in positions 338 and 341 than i n positions 335 and 336 (Fig. 4.4.). The presence o f Thr at positions 338 and 341 has, however, been associated to a negative influence in the dihydroxylation o f ort/20-substituted congeners (20, 54). In this study, the levels o f depletion o f 2 , 2 ' - d i C l biphenyl by B P D O B 3 5 6 and BPDO119 were comparable to those o f B P D O L B 4 O O - B P D O B 3 5 6 and BPDO119 contain a Thr i n positions 338 and 341, whereas B P D O L B 4 O O contains an A s n and He, respectively. The current results are thus more  consistent with the structural data than previous studies. The replacement o f region III in BphAELB4oo by that o f BphAEKF707 produced a dioxygenase that combined the broad  51  substrate specificity o f B P D O L B W O with the ability o f 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 o f other amino acids i n the overall reactivity o f the enzyme. It should also be noted that a given mutation could have different effects on the structure/activity o f an enzyme when combined with others. A s stated earlier, the combination o f 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 i n this study. The only way to definitively establish the role o f particular residues is by determining the crystal structure o f the variant protein. The superposition o f the active sites o f 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 o f BPDO119 is slightly larger. Although little structural data o f the relevant enzyme:congener complexes exists at this time, docking and minimization results indicate that B P D O 3 5 6 readily accommodates 3,3-' and 2,2'B  d i C l biphenyls i n 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 o f other factors on the activity o f the enzymes against the congener. O n the other hand, B P D O L B 4 O O can accommodate 3,3'- and 2 , 2 ' - d i C l 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 o f these congeners i n a catalytically competent configuration, despite the lower congener-transforming o f this variant with respect to B P D O B 3 5 6 - Based on docking and minimization experiments, BPDO119 binds 2 , 2 ' - d i C l and 2,2',5,5'-tetraCl biphenyl in a similar fashion to that o f B P D O L B 4 O O , whereas binding o f 3,3'-diCl biphenyl is similar to that o f B P D 0 B 5 6 - Thus, BPDO119 is 3  probably capable o f catalyzing the 3,4-dihydroxylation o f 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 i C l biphenyl. It is clear that the orientation o f the substrate i n the active site is strongly influenced by its chlorination pattern and the interactions o f the chlorines with specific residues o f the enzyme. Accordingly, a given amino acid substitution w i l l have different effects on the regiospecificity o f different congeners.  52  Considerable effort has been invested in engineering B P D O i n the last years. Most o f this effort involved, however, mutations o f residues i n region III. Although these play an important role i n the substrate preference o f the enzyme, it is possible that some other, as yet unidentified, residues play an equally important role. The publication o f the crystal structures o f BphAERHAi and other ring-hydroxylating dioxygenases should not only help to identify some o f these residues but should also provide a better understanding o f the role o f the previously identified residues and the mechanism.  The upcoming publication o f the  crystal structures  of  enzyme's  BphAE 356, B  BphAELB4oo and BphAEng is o f particular interest since they were obtained from highly active enzymes. These are also the first structures obtained from an engineered version o f B P D O , and comparison with the w i l d type enzyme should allow for the thorough investigation o f the role o f the mutated residues. Similarly, most protein engineering efforts have involved the use o f B P D O B 4 0 0 and B P D O K F 7 0 7 - The high PCB-transforming L  activity o f B P D O B 3 5 6 highlights the importance o f including this and other less well characterized enzymes i n 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 o f the enzyme. The engineered versions o f B P D O are rarely tested against other aromatic substrates.  Such testing may reveal some interesting novel  activities o f the enzyme.  53  5. BIBLIOGRAPHY 1.  2.  3.  4.  5.  6.  7.  8.  9.  10.  11.  Agar, N . Y. R. 2002. 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