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

Abstract

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 ϳ 2,3,4-trichloro > 3,3-dichloro > 2,4,4-trichloro > 4,4-dichloro ϳ 2,2-dichloro > 2,6-dichloro > 2,2,3,3-tetrachloro ϳ 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 ؋ 10 6 ؎ 0. ). 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-Å 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.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. 1) (46). The oxygenase (BphAE) has an ␣ 3 ␤ 3 composition. Each...

Biochemical Studies and Ligand-bound Structures of Biphenyl Dehydrogenase from Pandoraea pnomenusa Strain B-356 Reveal a Basis for Broad Specificity of the Enzyme

“…As expected from previous work with purified enzymes (12) and shown in Fig. 7, an isopropyl ␤-D-1-thiogalactopyranosideinduced resting cell suspension of E. coli harboring pDB31 .…”

“…Biochemical Analysis-It was reported by Gomez-Gil et al (12) that BPDO B-356 metabolizes 2,6-dichlorobiphenyl, 2,4,4Ј-trichlorobiphenyl, and 3,3Ј-dichlorobiphenyl to corresponding dihydrodiol metabolites more efficiently than BPDO LB400 . It is therefore interesting to examine the capacity of BphB B-356 to metabolize the dihydrodiol metabolites produced from these chlorobiphenyl congeners as they represent congeners with doubly ortho-, meta-, or para-substitution.…”

Roles of Ring-Hydroxylating Dioxygenases in Styrene and Benzene Catabolism inRhodococcus jostiiRHA1

“…Among characterized enzymes, BPDO shares the greatest amino acid sequence identity (98 to 99%) with toluene, isopropylbenzene, and benzene dioxygenases (34,43,50). Moreover, the substrate-binding pocket of the RHA1 enzyme (9) is not as large as that of other BPDOs, such as that of Pandoraea pnomenusa B-356 (11). By contrast, EBDO clusters with enzymes transforming larger substrates, such as phenanthrene dioxygenase from Burkholderia sp.…”

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

“…BphAE II9 is a variant of BphAE LB400 in which the seven residues of region III, 335 TFNNIRI 341 , are replaced by the corresponding residues of BphAE B356 , 333 GINTIRT 339 . Among other characteristics, BphAE II9 is able to transform 2,3,4=-trichlorobiphenyl more efficiently than is either parental enzyme (28). BphAE II10 adds a single-residue substitution to the BphAE II9 background: Ala267 is replaced by Ser, as occurs in BphAE B356 .…”