Characterization of Biphenyl Dioxygenase of Pandoraea pnomenusa B-356 As a Potent Polychlorinated Biphenyl-Degrading Enzyme

The microbial degradation of biphenyl has been well studied as a potential means of remediating soils contaminated with polychlorinated biphenyls (PCBs) (46). While the production of PCBs has been banned in industrial countries due to the adverse health effects that they cause in humans, these toxic pollutants are persistent and remain widespread in the environment (13). PCBs are aerobically transformed by the bph pathway, a pathway comprising four enzymes that initiates the catabolism of biphenyl. In most bacterial strains characterized to date, the pathway transforms up to tetrachlorobiphenyls, although some pathways can transform congeners containing up to six chlorine substituents (8, 44). A critical step in improving the microbial catabolic activities for the degradation of PCBs is understanding the reactivity of the four enzymes of the bph pathway for PCB metabolites. Biphenyl dioxygenase (BPDO), the first enzyme of the bph pathway, is a typical three-component, ring-hydroxylating dioxygenase that catalyzes the insertion of molecular oxygen into an aromatic ring, forming cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene (Fig. ​(Fig.1)1) (46). The oxygenase (BphAE) has an α3β3 composition. Each α subunit (BphA) contains a Rieske-type Fe2S2 cluster and a mononuclear iron center, located at the enzyme's active site. A reductase (BphG) and a ferredoxin (BphF) function to transfer electrons from NADH to the Rieske center of BphAE, where they are used in the hydroxylation of the biphenyl at the mononuclear iron center. The mechanism of dihydroxylation is thought to be very similar to that of the naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4, the best-characterized ring-hydroxylating dioxygenase (35). BPDO is a major determinant of a bacterium's PCB-transforming capabilities. Studies on BPDOs from different organisms have revealed significant differences in congener (substrate) preference and regiospecificity. For example, BPDOKF707 and BPDOLB00, from Pseudomonas alcaligenes KF707 and Burkholderia xenovorans LB400, respectively, show very different reactivities, although they share more than 95% sequence identity (21, 55). BPDOLB400 preferentially transforms ortho-substituted congeners containing up to six chlorines (10, 43). This enzyme has the relatively unusual ability to catalyze the 3,4-dihydroxylation of certain 2,5-substituted congeners, such as 2,2′,5,5′-tetrachlorobiphenyl (32). BPDOLB400 is also remarkable in that it catalyzes the dehalogenation of certain 2-Cl congeners, yielding 2,3-dihydroxybiphenyls (25, 50-52), the substrate of the third Bph pathway enzyme. By contrast, BPDOKF707 transforms a narrow range of PCB congeners and catalyzes neither 3,4-dihydroxylation nor o-dechlorination. Moreover, it preferentially transforms 4,4′-dichlorobiphenyl over either 3,3′- or 2,2′-dichlorobiphenyl (24). A third enzyme, BPDOB356 from Pandoraea pnomenusa (formerly Comamonas testosteroni [59]) B356, shares approximately 70% sequence identity with both BPDOKF707 and BPDOLB400. Previous studies have indicated this enzyme's preference for meta- > ortho- > para-substituted dichlorobiphenyls (33), as well as its higher specific activity against biphenyl than that of BPDOLB00. FIG. 1. Dihydroxylation of biphenyl by BPDO. The enzyme comprises a flavin adenine dinucleotide-containing reductase (BphG), a Rieske-type ferredoxin (BphF), and an oxygenase that contains a Rieske-type Fe2S2 cluster and a catalytic mononuclear iron center. Biphenyl ... Protein engineering efforts have yielded BPDO with improved PCB-degrading capabilities, as well as insights into the molecular basis of congener preference. Mutagenesis studies of BphALB400 and BphAKF707 identified four regions (regions I to IV) whose sequences influence the range of congeners attacked (44). All four regions occur in the C-terminal domain of BphA, consistent with the location of the oxygenase's active site. Substitution of individual residues in region III of BphALB400, comprising Thr335, Phe336, Asn338, and Ile341, improved the ability of the enzyme to transform 4,4′-dichlorobiphenyls, although the greatest improvements in activity were achieved by multiple substitutions in this region, suggesting that there is a cooperative or additive effect (4). Several studies have since confirmed the importance of these residues in determining the enzyme's congener preference and regiospecificity (3-5, 36, 54). For example, BphAEII9, a variant of BphAELB400 containing region III of BphAB356, is reported to possess better PCB-degrading properties than either parental enzyme (3). By contrast, BphAEII10, which differs from BphAEII9 by a single residue at position 267 (Ser, as in BphAEB356, instead of Ala, as in BphAELB400), was not able to transform any of the PCBs tested. Here, we report the characterization of four variants of BPDO: BphAELB400, BphAEB356, and two variants of these enzymes generated via directed evolution, BphAEII9 and BphAEII10. The steady-state kinetic parameters for biphenyl of anaerobically purified non-His-tagged enzymes were determined, and the activities towards various PCB congeners were investigated. The degree of uncoupling between O2 utilization and congener transformation was also determined for the different enzymes using different congeners. To validate previous studies, activities determined using purified enzymes and whole cells were compared, as were activities determined using individual congeners and mixtures of congeners. Finally, a crystal structure of the BphAEB356-2,6-dichlorobiphenyl complex was determined. The results are discussed in terms of the proposed catalytic mechanism of ring-hydroxylating dioxygenases and the specificities of the different BPDOs.