Engineering Burkholderia xenovorans LB400 BphA through Site-Directed Mutagenesis at Position 283.

Biphenyl dioxygenase (BPDO), which is a Rieske-type oxygenase (RO), catalyzes the initial dioxygenation of biphenyl and some polychlorinated biphenyls (PCBs). In order to enhance the degradation ability of BPDO in terms of a broader substrate range, the BphAES283M, BphAEp4-S283M, and BphAERR41-S283M variants were created from the parent enzymes BphAELB400, BphAEp4, and BphAERR41, respectively, by a substitution at one residue, Ser283Met. The results of steady-state kinetic parameters show that for biphenyl, the kcat/Km values of BphAES283M, BphAEp4-S283M, and BphAERR41-S283M were significantly increased compared to those of their parent enzymes. Meanwhile, we determined the steady-state kinetics of BphAEs toward highly chlorinated biphenyls. The results suggested that the Ser283Met substitution enhanced the catalytic activity of BphAEs toward 2,3',4,4'-tetrachlorobiphenyl (2,3',4,4'-CB), 2,2',6,6'-tetrachlorobiphenyl (2,2',6,6'-CB), and 2,3',4,4',5-pentachlorobiphenyl (2,3',4,4',5-CB). We compared the catalytic reactions of BphAELB400 and its variants toward 2,2'-dichlorobiphenyl (2,2'-CB), 2,5-dichlorobiphenyl (2,5-CB), and 2,6-dichlorobiphenyl (2,6-CB). The biochemical data indicate that the Ser283Met substitution alters the orientation of the substrate inside the catalytic site and, thereby, its site of hydroxylation, and this was confirmed by docking experiments. We also assessed the substrate ranges of BphAELB400 and its variants with degradation activity. BphAES283M and BphAEp4-S283M were clearly improved in oxidizing some of the 3-6-chlorinated biphenyls, which are generally very poorly oxidized by most dioxygenases. Collectively, the present work showed a significant effect of mutation Ser283Met on substrate specificity/regiospecificity in BPDO. These will certainly be meaningful elements for understanding the effect of the residue corresponding to position 283 in other Rieske oxygenase enzymes.IMPORTANCE The segment from positions 280 to 283 in BphAEs is located at the entrance of the catalytic pocket, and it shows variation in conformation. In previous works, results have suggested but never proved that residue Ser283 of BphAELB400 might play a role in substrate specificity. In the present paper, we found that the Ser283Met substitution significantly increased the specificity of the reaction of BphAE toward biphenyl, 2,3',4,4'-CB, 2,2',6,6'-CB, and 2,3',4,4',5-CB. Meanwhile, the Ser283Met substitution altered the regiospecificity of BphAE toward 2,2'-dichlorobiphenyl and 2,6-dichlorobiphenyl. Additionally, this substitution extended the range of PCBs metabolized by the mutated BphAE. BphAES283M and BphAEp4-S283M were clearly improved in oxidizing some of the more highly chlorinated biphenyls (3 to 6 chlorines), which are generally very poorly oxidized by most dioxygenases. We used modeled and docked enzymes to identify some of the structural features that explain the new properties of the mutant enzymes. Altogether, the results of this study provide better insights into the mechanisms by which BPDO evolves to change and/or expand its substrate range and its regiospecificity.

Structural Basis of the Enhanced Pollutant-Degrading Capabilities of an Engineered Biphenyl Dioxygenase.

UNLABELLED: Biphenyl dioxygenase, the first enzyme of the biphenyl catabolic pathway, is a major determinant of which polychlorinated biphenyl (PCB) congeners are metabolized by a given bacterial strain. Ongoing efforts aim to engineer BphAE, the oxygenase component of the enzyme, to efficiently transform a wider range of congeners. BphAEII9, a variant of BphAELB400 in which a seven-residue segment, (335)TFNNIRI(341), has been replaced by the corresponding segment of BphAEB356, (333)GINTIRT(339), transforms a broader range of PCB congeners than does either BphAELB400 or BphAEB356, including 2,6-dichlorobiphenyl, 3,3'-dichlorobiphenyl, 4,4'-dichlorobiphenyl, and 2,3,4'-trichlorobiphenyl. To understand the structural basis of the enhanced activity of BphAEII9, we have determined the three-dimensional structure of this variant in substrate-free and biphenyl-bound forms. Structural comparison with BphAELB400 reveals a flexible active-site mouth and a relaxed substrate binding pocket in BphAEII9 that allow it to bind different congeners and which could be responsible for the enzyme's altered specificity. Biochemical experiments revealed that BphAEII9 transformed 2,3,4'-trichlorobiphenyl and 2,2',5,5'-tetrachlorobiphenyl more efficiently than did BphAELB400 and BphAEB356 BphAEII9 also transformed the insecticide dichlorodiphenyltrichloroethane (DDT) more efficiently than did either parental enzyme (apparent kcat/Km of 2.2 ± 0.5 mM(-1) s(-1), versus 0.9 ± 0.5 mM(-1) s(-1) for BphAEB356). Studies of docking of the enzymes with these three substrates provide insight into the structural basis of the different substrate selectivities and regiospecificities of the enzymes. IMPORTANCE: Biphenyl dioxygenase is the first enzyme of the biphenyl degradation pathway that is involved in the degradation of polychlorinated biphenyls. Attempts have been made to identify the residues that influence the enzyme activity for the range of substrates among various species. In this study, we have done a structural study of one variant of this enzyme that was produced by family shuffling of genes from two different species. Comparison of the structure of this variant with those of the parent enzymes provided an important insight into the molecular basis for the broader substrate preference of this enzyme. The structural and functional details gained in this study can be utilized to further engineer desired enzymatic activity, producing more potent enzymes.

Optimizing Polychlorinated Biphenyl Degradation by Flavonoid-Induced Cells of the Rhizobacterium Rhodococcus erythropolis U23A.

There is evidence that many plant secondary metabolites may act as signal molecules to trigger the bacterial ability to metabolize polychlorinated biphenyls (PCBs) during the rhizoremediation process. However, the bases for the PCB rhizoremediation process are still largely unknown. The rhizobacterium Rhodococcus erythropolis U23A is unable to use flavanone as a growth substrate. However, on the basis of an assay that monitors the amount of 4-chlorobenzoate produced from 4-chlorobiphenyl by cells grown co-metabolically on flavanone plus sodium acetate, this flavonoid was previously found to be a potential inducer of the U23A biphenyl catabolic pathway. In this work, and using the same assay, we identified ten other flavonoids that did not support growth, but that acted as inducers of the U23A biphenyl pathway, and we confirmed flavonoid induction of the biphenyl catabolic pathway using quantitative real-time polymerase chain reaction (RT-qPCR) on the bphA gene. We also examined the effect of the growth co-substrate on flavonoid induction. Sodium acetate was replaced by glucose, mannose, sucrose, or mannitol, which are sugars found in plant root exudates. The data showed that the level of induction of strain U23A biphenyl-degrading enzymes was significantly influenced by the nature and concentration of the flavonoid in the growth medium, as well as by the substrate used for growth. Sucrose allowed for an optimal induction response for most flavonoids. Some flavonoids, such as flavone and isoflavone, were better inducers of the biphenyl catabolic enzymes than biphenyl itself. We also found that all flavonoids tested in this work were metabolized by strain U23A during co-metabolic growth, but that the metabolite profiles, as well as the level of efficiency of degradation, differed for each flavonoid. To obtain insight into how flavonoids interact with strain U23A to promote polychlorinated biphenyl (PCB) degradation, we determined the concentration of flavanone at which optimal PCB-degrading performance of strain U23A was achieved. We showed that it corresponded to the concentration required to fully induce the biphenyl catabolic pathway of the strain. Together, our data demonstrate that optimal PCB degradation during the rhizoremediation process will require the adjustment of several parameters, including the presence of the appropriate flavonoids at the proper concentrations and the presence of proper growth substrates that positively influence the ability of flavonoids to induce the pathway.

Metabolism of Doubly para-Substituted Hydroxychlorobiphenyls by Bacterial Biphenyl Dioxygenases.

In this work, we examined the profile of metabolites produced from the doubly para-substituted biphenyl analogs 4,4'-dihydroxybiphenyl, 4-hydroxy-4'-chlorobiphenyl, 3-hydroxy-4,4'-dichlorobiphenyl, and 3,3'-dihydroxy-4,4'-chlorobiphenyl by biphenyl-induced Pandoraea pnomenusa B356 and by its biphenyl dioxygenase (BPDO). 4-Hydroxy-4'-chlorobiphenyl was hydroxylated principally through a 2,3-dioxygenation of the hydroxylated ring to generate 2,3-dihydro-2,3,4-trihydroxy-4'-chlorobiphenyl and 3,4-dihydroxy-4'-chlorobiphenyl after the removal of water. The former was further oxidized by the biphenyl dioxygenase to produce ultimately 3,4,5-trihydroxy-4'-chlorobiphenyl, a dead-end metabolite. 3-Hydroxy-4,4'-dichlorobiphenyl was oxygenated on both rings. Hydroxylation of the nonhydroxylated ring generated 2,3,3'-trihydroxy-4'-chlorobiphenyl with concomitant dechlorination, and 2,3,3'-trihydroxy-4'-chlorobiphenyl was ultimately metabolized to 2-hydroxy-4-chlorobenzoate, but hydroxylation of the hydroxylated ring generated dead-end metabolites. 3,3'-Dihydroxy-4,4'-dichlorobiphenyl was principally metabolized through a 2,3-dioxygenation to generate 2,3-dihydro-2,3,3'-trihydroxy-4,4'-dichlorobiphenyl, which was ultimately converted to 3-hydroxy-4-chlorobenzoate. Similar metabolites were produced when the biphenyl dioxygenase of Burkholderia xenovorans LB400 was used to catalyze the reactions, except that for the three substrates used, the BPDO of LB400 was less efficient than that of B356, and unlike that of B356, it was unable to further oxidize the initial reaction products. Together the data show that BPDO oxidation of doubly para-substituted hydroxychlorobiphenyls may generate nonnegligible amounts of dead-end metabolites. Therefore, biphenyl dioxygenase could produce metabolites other than those expected, corresponding to dihydrodihydroxy metabolites from initial doubly para-substituted substrates. This finding shows that a clear picture of the fate of polychlorinated biphenyls in contaminated sites will require more insights into the bacterial metabolism of hydroxychlorobiphenyls and the chemistry of the dihydrodihydroxylated metabolites derived from them.

Engineering a thermostable fungal GH10 xylanase, importance of N-terminal amino acids.

Xylanases are used in many industrial processes including pulp bleaching, baking, detergent, and the hydrolysis of plant cell wall in biofuels production. In this work we have evolved a single domain GH10 xylanase, Xyn10A_ASPNG, from Aspergillus niger to improve its thermostability. We introduced a rational approach involving as the first step a computational analysis to guide the design of a mutagenesis library in targeted regions which identified thermal important residues that were subsequently randomly mutagenized through rounds of iterative saturation mutagenesis (ISM). Focusing on five residues, four rounds of ISM had generated a quintuple mutant 4S1 (R25W/V29A/I31L/L43F/T58I) which exhibited thermal inactivation half-life (t1/2 ) at 60°C that was prolonged by 30 folds in comparison with wild-type enzyme. Whereas the wild-type enzyme retained 0.2% of its initial activity after a heat treatment of 10 min at 60°C and was completely inactivated after 2 min at 65°C, 4S1 mutant retained 30% of its initial activity after 15 min heating at 65°C. Furthermore, the mutant melting temperature (Tm ) increased by 17.4°C compared to the wild type. Each of the five mutations in 4S1 was found to contribute to thermoresistance, but the dramatic improvement of enzyme thermoresistance of 4S1 was attributed to the synergistic effects of the five mutations. Comparison of biochemical data and model structure between 4S1 and the wild-type enzyme suggested that the N-terminal coil of the enzyme is important in stabilizing GH10 xylanase structure. Based on model structure analyses, we propose that enforced hydrophobic interactions within N-terminal elements and between N- and C-terminal ends are responsible for the improved thermostability of Xyn10A_ASPNG.

Has the bacterial biphenyl catabolic pathway evolved primarily to degrade biphenyl? The diphenylmethane case.

In this work, we have compared the ability of Pandoraea pnomenusa B356 and of Burkholderia xenovorans LB400 to metabolize diphenylmethane and benzophenone, two biphenyl analogs in which the phenyl rings are bonded to a single carbon. Both chemicals are of environmental concern. P. pnomenusa B356 grew well on diphenylmethane. On the basis of growth kinetics analyses, diphenylmethane and biphenyl were shown to induce the same catabolic pathway. The profile of metabolites produced during growth of strain B356 on diphenylmethane was the same as the one produced by isolated enzymes of the biphenyl catabolic pathway acting individually or in coupled reactions. The biphenyl dioxygenase oxidizes diphenylmethane to 3-benzylcyclohexa-3,5-diene-1,2-diol very efficiently, and ultimately this metabolite is transformed to phenylacetic acid, which is further metabolized by a lower pathway. Strain B356 was also able to cometabolize benzophenone through its biphenyl pathway, although in this case, this substrate was unable to induce the biphenyl catabolic pathway and the degradation was incomplete, with accumulation of 2-hydroxy-6,7-dioxo-7-phenylheptanoic acid. Unlike strain B356, B. xenovorans LB400 did not grow on diphenylmethane. Its biphenyl pathway enzymes metabolized diphenylmethane, but they poorly metabolize benzophenone. The fact that the biphenyl catabolic pathway of strain B356 metabolized diphenylmethane and benzophenone more efficiently than that of strain LB400 brings us to postulate that in strain B356, this pathway evolved divergently to serve other functions not related to biphenyl degradation.

Prospects for using combined engineered bacterial enzymes and plant systems to rhizoremediate polychlorinated biphenyls.

The fate of polychlorinated biphenyls (PCBs) in soil is driven by a combination of interacting biological processes. Several investigations have brought evidence that the rhizosphere provides a remarkable ecological niche to enhance the PCB degradation process by rhizobacteria. The bacterial oxidative enzymes involved in PCB degradation have been investigated extensively and novel engineered enzymes exhibiting enhanced catalytic activities toward more persistent PCBs have been described. Furthermore, recent studies suggest that approaches involving processes based on plant-microbe associations are very promising to remediate PCB-contaminated sites. In this review emphasis will be placed on the current state of knowledge regarding the strategies that are proposed to engineer the enzymes of the PCB-degrading bacterial oxidative pathway and to design PCB-degrading plant-microbe systems to remediate PCB-contaminated soil.

Structural insights into the metabolism of 2-chlorodibenzofuran by an evolved biphenyl dioxygenase.

The biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE(LB400)) is a Rieske-type oxygenase that catalyzes the stereospecific oxygenation of many heterocyclic aromatics including dibenzofuran. In a previous work, we evolved BphAE(LB400) and obtained BphAE(RR41). This variant metabolizes dibenzofuran and 2-chlorodibenzofuran more efficiently than BphAE(LB400). However, the regiospecificity of BphAE(RR41) toward these substrates differs. Dibenzofuran is metabolized principally through a lateral dioxygenation whereas 2-chlorodibenzofuran is metabolized principally through an angular dioxygenation. In order to explain this difference, we examined the crystal structures of both substrate-bound forms of BphAE(RR41) obtained under anaerobic conditions. This structure analysis, in combination with biochemical data for a Ser283Gly mutant provided evidences that the substrate is compelled to move after oxygen-binding in BphAE(RR41):dibenzofuran. In BphAE(RR41):2-chlorodibenzofuran, the chlorine atom is close to the side chain of Ser283. This contact is missing in the BphAE(RR41):dibenzofuran, and strong enough in the BphAE(RR41):2-chlorodibenzofuran to help prevent substrate movement during the catalytic reaction.

Remarkable ability of Pandoraea pnomenusa B356 biphenyl dioxygenase to metabolize simple flavonoids.

Many investigations have provided evidence that plant secondary metabolites, especially flavonoids, may serve as signal molecules to trigger the abilities of bacteria to degrade chlorobiphenyls in soil. However, the bases for this interaction are largely unknown. In this work, we found that BphAE(B356), the biphenyl/chlorobiphenyl dioxygenase from Pandoraea pnomenusa B356, is significantly better fitted to metabolize flavone, isoflavone, and flavanone than BphAE(LB400) from Burkholderia xenovorans LB400. Unlike those of BphAE(LB400), the kinetic parameters of BphAE(B356) toward these flavonoids were in the same range as for biphenyl. In addition, remarkably, the biphenyl catabolic pathway of strain B356 was strongly induced by isoflavone, whereas none of the three flavonoids induced the catabolic pathway of strain LB400. Docking experiments that replaced biphenyl in the biphenyl-bound form of the enzymes with flavone, isoflavone, or flavanone showed that the superior ability of BphAE(B356) over BphAE(LB400) is principally attributable to the replacement of Phe336 of BphAE(LB400) by Ile334 and of Thr335 of BphAE(LB400) by Gly333 of BphAE(B356). However, biochemical and structural comparison of BphAE(B356) with BphAE(p4), a mutant of BphAE(LB400) which was obtained in a previous work by the double substitution Phe336Met Thr335Ala of BphAE(LB400), provided evidence that other residues or structural features of BphAE(B356) whose precise identification the docking experiment did not allow are also responsible for the superior catalytic abilities of BphAE(B356). Together, these data provide supporting evidence that the biphenyl catabolic pathways have evolved divergently among proteobacteria, where some of them may serve ecological functions related to the metabolism of plant secondary metabolites in soil.

Metabolism of chlorobiphenyls by a variant biphenyl dioxygenase exhibiting enhanced activity toward dibenzofuran.

The biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE(LB400)) catalyzes the dihydroxylation of biphenyl and of several polychlorinated biphenyls (PCBs) but it poorly oxidizes dibenzofuran. In this work we showed that BphAE(RR41), a variant which was previously found to metabolize dibenzofuran more efficiently than its parent BphAE(LB400), metabolized a broader range of PCBs than BphAE(LB400). Hence, BphAE(RR41) was able to metabolize 2,6,2',6'-, 3,4,3',5'- and 2,4,3',4'-tetrachlorobiphenyl that BphAE(LB400) is unable to metabolize. BphAE(RR41) was obtained by changing Thr335Phe336Asn338Ile341Leu409 of BphAE(LB400) to Ala335Met336Gln338Val341Phe409. Site-directed mutagenesis was used to create combinations of each substitution, in order to assess their individual contributions. Data show that the same Asn338Glu/Leu409Phe substitution that enhanced the ability to metabolize dibenzofuran resulted in a broadening of the PCB substrates range of the enzyme. The role of these substitutions on regiospecificities toward selected PCBs is also discussed.

Plant exudates promote PCB degradation by a rhodococcal rhizobacteria.

Rhodococcus erythropolis U23A is a polychlorinated biphenyl (PCB)-degrading bacterium isolated from the rhizosphere of plants grown on a PCB-contaminated soil. Strain U23A bphA exhibited 99% identity with bphA1 of Rhodococcus globerulus P6. We grew Arabidopsis thaliana in a hydroponic axenic system, collected, and concentrated the plant secondary metabolite-containing root exudates. Strain U23A exhibited a chemotactic response toward these root exudates. In a root colonizing assay, the number of cells of strain U23A associated to the plant roots (5.7 × 105 CFU g⁻¹) was greater than the number remaining in the surrounding sand (4.5 × 104 CFU g⁻¹). Furthermore, the exudates could support the growth of strain U23A. In a resting cell suspension assay, cells grown in a minimal medium containing Arabidopsis root exudates as sole growth substrate were able to metabolize 2,3,4'- and 2,3',4-trichlorobiphenyl. However, no significant degradation of any of congeners was observed for control cells grown on Luria-Bertani medium. Although strain U23A was unable to grow on any of the flavonoids identified in root exudates, biphenyl-induced cells metabolized flavanone, one of the major root exudate components. In addition, when used as co-substrate with sodium acetate, flavanone was as efficient as biphenyl to induce the biphenyl catabolic pathway of strain U23A. Together, these data provide supporting evidence that some rhodococci can live in soil in close association with plant roots and that root exudates can support their growth and trigger their PCB-degrading ability. This suggests that, like the flagellated Gram-negative bacteria, non-flagellated rhodococci may also play a key role in the degradation of persistent pollutants.

Insight into the metabolism of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by biphenyl dioxygenases.

In this work we have investigated the ability of the biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE(LB400)) and of Pandoraea pnomenusa B356 (BphAE(B356)) to metabolize DDT. Data show BphAE(LB400) is unable to metabolize this substrate but BphAE(B356) metabolizes DDT to produce two stereoisomers. Structural analysis of DDT-docked BphAE(LB400) and BphAE(B356) identified residue Phe336 of BphAE(LB400) as critical to prevent productive binding of DDT to BphAE(LB400). Furthermore, the fact that residue Gly319 of BphAE(B356) is less constrained than Gly321 of BphAE(LB400) most likely contributes to the ability of BphAE(B356) to bind DDT productively. This was confirmed by examining the ability of BphAE chimeras obtained by shuffling bphA genes from strain B356 and LB400. Chimeras where residues Thr335 (which modulates the constraints on Gly321) and Phe336 (which contacts the substrate) of BphAE(LB400) were replaced by Gly and Ile respectively were able to metabolize DDT. However their stereospecificities varied depending on the presence of other segments or residues from BphAE(B356). Structural analysis suggests that either one or both of residue 267 and a segments comprised of residue 247-260 are likely involved in stereospecificity.

Biochemical studies and ligand-bound structures of biphenyl dehydrogenase from Pandoraea pnomenusa strain B-356 reveal a basis for broad specificity of the enzyme.

Biphenyl dehydrogenase, a member of short-chain dehydrogenase/reductase enzymes, catalyzes the second step of the biphenyl/polychlorinated biphenyls catabolic pathway in bacteria. To understand the molecular basis for the broad substrate specificity of Pandoraea pnomenusa strain B-356 biphenyl dehydrogenase (BphB(B-356)), the crystal structures of the apo-enzyme, the binary complex with NAD(+), and the ternary complexes with NAD(+)-2,3-dihydroxybiphenyl and NAD(+)-4,4'-dihydroxybiphenyl were determined at 2.2-, 2.5-, 2.4-, and 2.1-Å resolutions, respectively. A crystal structure representing an intermediate state of the enzyme was also obtained in which the substrate binding loop was ordered as compared with the apo and binary forms but it was displaced significantly with respect to the ternary structures. These five structures reveal that the substrate binding loop is highly mobile and that its conformation changes during ligand binding, starting from a disorganized loop in the apo state to a well organized loop structure in the ligand-bound form. Conformational changes are induced during ligand binding; forming a well defined cavity to accommodate a wide variety of substrates. This explains the biochemical data that shows BphB(B-356) converts the dihydrodiol metabolites of 3,3'-dichlorobiphenyl, 2,4,4'-trichlorobiphenyl, and 2,6-dichlorobiphenyl to their respective dihydroxy metabolites. For the first time, a combination of structural, biochemical, and molecular docking studies of BphB(B-356) elucidate the unique ability of the enzyme to transform the cis-dihydrodiols of double meta-, para-, and ortho-substituted chlorobiphenyls.

Retuning Rieske-type oxygenases to expand substrate range.

Rieske-type oxygenases are promising biocatalysts for the destruction of persistent pollutants or for the synthesis of fine chemicals. In this work, we explored pathways through which Rieske-type oxygenases evolve to expand their substrate range. BphAE(p4), a variant biphenyl dioxygenase generated from Burkholderia xenovorans LB400 BphAE(LB400) by the double substitution T335A/F336M, and BphAE(RR41), obtained by changing Asn(338), Ile(341), and Leu(409) of BphAE(p4) to Gln(338), Val(341), and Phe(409), metabolize dibenzofuran two and three times faster than BphAE(LB400), respectively. Steady-state kinetic measurements of single- and multiple-substitution mutants of BphAE(LB400) showed that the single T335A and the double N338Q/L409F substitutions contribute significantly to enhanced catalytic activity toward dibenzofuran. Analysis of crystal structures showed that the T335A substitution relieves constraints on a segment lining the catalytic cavity, allowing a significant displacement in response to dibenzofuran binding. The combined N338Q/L409F substitutions alter substrate-induced conformational changes of protein groups involved in subunit assembly and in the chemical steps of the reaction. This suggests a responsive induced fit mechanism that retunes the alignment of protein atoms involved in the chemical steps of the reaction. These enzymes can thus expand their substrate range through mutations that alter the constraints or plasticity of the catalytic cavity to accommodate new substrates or that alter the induced fit mechanism required to achieve proper alignment of reaction-critical atoms or groups.

Anaerobic crystallization and initial X-ray diffraction data of biphenyl 2,3-dioxygenase from Burkholderia xenovorans LB400: addition of agarose improved the quality of the crystals.

Biphenyl 2,3-dioxygenase (BPDO; EC 1.14.12.18) catalyzes the initial step in the degradation of biphenyl and some polychlorinated biphenyls (PCBs). BPDOLB400, the terminal dioxygenase component from Burkholderia xenovorans LB400, a proteobacterial species that degrades a broad range of PCBs, has been crystallized under anaerobic conditions by sitting-drop vapour diffusion. Initial crystals obtained using various polyethylene glycols as precipitating agents diffracted to very low resolution (∼8 Å) and the recorded reflections were diffuse and poorly shaped. The quality of the crystals was significantly improved by the addition of 0.2% agarose to the crystallization cocktail. In the presence of agarose, wild-type BPDOLB400 crystals that diffracted to 2.4 Šresolution grew in space group P1. Crystals of the BPDOP4 and BPDORR41 variants of BPDOLB400 grew in space group P2(1).

Structural insight into the expanded PCB-degrading abilities of a biphenyl dioxygenase obtained by directed evolution.

The biphenyl dioxygenase of Burkholderia xenovorans LB400 is a multicomponent Rieske-type oxygenase that catalyzes the dihydroxylation of biphenyl and many polychlorinated biphenyls (PCBs). The structural bases for the substrate specificity of the enzyme's oxygenase component (BphAE(LB400)) are largely unknown. BphAE(p4), a variant previously obtained through directed evolution, transforms several chlorobiphenyls, including 2,6-dichlorobiphenyl, more efficiently than BphAE(LB400), yet differs from the parent oxygenase at only two positions: T335A/F336M. Here, we compare the structures of BphAE(LB400) and BphAE(p4) and examine the biochemical properties of two BphAE(LB400) variants with single substitutions, T335A or F336M. Our data show that residue 336 contacts the biphenyl and influences the regiospecificity of the reaction, but does not enhance the enzyme's reactivity toward 2,6-dichlorobiphenyl. By contrast, residue 335 does not contact biphenyl but contributes significantly to expansion of the enzyme's substrate range. Crystal structures indicate that Thr335 imposes constraints through hydrogen bonds and nonbonded contacts to the segment from Val320 to Gln322. These contacts are lost when Thr is replaced by Ala, relieving intramolecular constraints and allowing for significant movement of this segment during binding of 2,6-dichlorobiphenyl, which increases the space available to accommodate the doubly ortho-chlorinated congener 2,6-dichlorobiphenyl. This study provides important insight about how Rieske-type oxygenases can expand substrate range through mutations that increase the plasticity and/or mobility of protein segments lining the catalytic cavity.

Expression, purification, crystallization and preliminary crystallographic studies of cis-biphenyl-2,3-dihydrodiol-2,3-dehydrogenase from Pandoraea pnomenusa B-356.

cis-Biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (BphB) is involved in the aerobic biodegradation of biphenyl and polychlorinated biphenyls. BphB from Pandoraea pnomenusa strain B-356 was overexpressed in Escherichia coli, purified to homogeneity and crystallized. Crystals were obtained by the sitting-drop vapour-diffusion method using polyethylene glycol 3350 and 0.2 M sodium malonate. A BphB crystal diffracted to 2.8 Šresolution and belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 75.2, c = 180.4 Å. Preliminary crystallographic analysis indicated the presence of two molecules in the asymmetric unit, giving a Matthews coefficient of 2.2 Å(3) Da(-1) and a solvent content of 44%.

Transgenic plants to improve rhizoremediation of polychlorinated biphenyls (PCBs).

Recent investigations have shown that the three components of the biphenyl dioxygenase and the 2,3-dihydroxybiphenyl dioxygenase can be produced actively in transgenic plants. Both enzymes catalyze critical steps of the bacterial polychlorinated biphenyl (PCB) degrading pathway. On the basis of these observations, optimized plant-microbe bioremediation processes in which transgenic plants would initiate PCB metabolism and release the metabolites for further degradation by rhizobacteria has been proposed. Since this is still a relatively new approach for PCB remediation, its successful application will require efforts first, to engineer improved PCB-degrading enzymes; second, to co-ordinately express these enzymes' components in plants; and third, to better understand the mechanisms by which plants and rhizobacteria interact to degrade organic pollutants.

Diversity of the C-terminal portion of the biphenyl dioxygenase large subunit.

The biphenyl dioxygenase (BPDO) catalyses a stereospecific dioxygenation of biphenyl and analogs of it. Aside from being involved in the destruction and detoxification of toxic pollutants in soil, in the context of the green chemistry concept, this enzyme is a promising biocatalyst to design new more selective and more environmentally friendly approaches to manufacture fine chemicals. At this time, most of our knowledge about the variability of key residues determining the substrate specificity and regiospecificity of the enzyme oxygenase component (BphAE) toward biphenyl analogs and about the effect of altering these residues on catalytic properties is based on investigations made with BphAEs from cultured organisms and engineered enzymes derived from them. The purpose of this work was to examine the diversity of the amino acid sequence patterns of the alpha subunit (BphA) C-terminal domain deduced from PCR products amplified from DNA extracted from cultured bacteria of various phylogenetic lines and from the soil microflora of PCB-contaminated soils. Of special interest were segments of the C-terminal portion called regions I, III and IV. Altogether, the phylogenetic tree obtained from aligning the deduced amino acid sequences of BphAs C-terminal domain from cultured bacteria belonging to various ecological niches and from uncultured soil bacteria reveals that most of the BphAs were linked to the three clusters of BphAs previously reported. However, few belong to new branches that diverge from the previously known branches showing a high diversity of BphAs in natural environment. Furthermore, data show a wide distribution of BphAs with family linkages that not only crosses bacterial taxonomic frontiers but also ecological niches. Nevertheless, in spite of this divergence, the sequence patterns of regions III and IV amino acids that are known to influence substrate specificity and regiospecificity are rather conserved among BphAs and the pattern was independent of the family cluster to which they belong. In most cases, regions III and IV amino acid patterns are closer to those of Pseudomonas pseudoalcaligenes KF707 BphA1 than to the most versatile Burkholderia xenovorans LB400 BphA. This might suggest that the PCB-degrading potency of soil bacteria is closer to the one observed for KF707 BphAE than from LB400 BphAE. However, the fact that among less than 20 PCR products amplified from soil DNA that we have sequenced, one of them was very homologous to that of LB400 BphA and in addition, residues 335 and 336 of LB400 were replaced by residues that previous enzyme engineering had shown to extend the range of PCB substrate used by the enzyme strongly suggest that PCB-degrading bacteria are evolving in soil to optimize their PCB-degrading capacity.

Characterization of biphenyl dioxygenase of Pandoraea pnomenusa B-356 as a potent polychlorinated biphenyl-degrading enzyme.

Biphenyl dioxygenase (BPDO) catalyzes the aerobic transformation of biphenyl and various polychlorinated biphenyls (PCBs). In three different assays, BPDO(B356) from Pandoraea pnomenusa B-356 was a more potent PCB-degrading enzyme than BPDO(LB400) from Burkholderia xenovorans LB400 (75% amino acid sequence identity), transforming nine congeners in the following order of preference: 2,3',4-trichloro approximately 2,3,4'-trichloro > 3,3'-dichloro > 2,4,4'-trichloro > 4,4'-dichloro approximately 2,2'-dichloro > 2,6-dichloro > 2,2',3,3'-tetrachloro approximately 2,2',5,5'-tetrachloro. Except for 2,2',5,5'-tetrachlorobiphenyl, BPDO(B356) transformed each congener at a higher rate than BPDO(LB400). The assays used either whole cells or purified enzymes and either individual congeners or mixtures of congeners. Product analyses established previously unrecognized BPDO(B356) activities, including the 3,4-dihydroxylation of 2,6-dichlorobiphenyl. BPDO(LB400) had a greater apparent specificity for biphenyl than BPDO(B356) (k(cat)/K(m) = 2.4 x 10(6) +/- 0.7 x 10(6) M(-1) s(-1) versus k(cat)/K(m) = 0.21 x 10(6) +/- 0.04 x 10(6) M(-1) s(-1)). However, the latter transformed biphenyl at a higher maximal rate (k(cat) = 4.1 +/- 0.2 s(-1) versus k(cat) = 0.4 +/- 0.1 s(-1)). A variant of BPDO(LB400) containing four active site residues of BPDO(B356) transformed para-substituted congeners better than BPDO(LB400). Interestingly, a substitution remote from the active site, A267S, increased the enzyme's preference for meta-substituted congeners. Moreover, this substitution had a greater effect on the kinetics of biphenyl utilization than substitutions in the substrate-binding pocket. In all variants, the degree of coupling between congener depletion and O(2) consumption was approximately proportional to congener depletion. At 2.4-A resolution, the crystal structure of the BPDO(B356)-2,6-dichlorobiphenyl complex, the first crystal structure of a BPDO-PCB complex, provided additional insight into the reactivity of this isozyme with this congener, as well as into the differences in congener preferences of the BPDOs.