Dataset Information

Genetic structures of the genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase from biphenyl- and 4-chlorobiphenyl-degrading Pseudomonas sp. strain DJ-12.

ABSTRACT: The pcbC and pcbD genes of Pseudomonas sp. strain DJ-12, a natural isolate degrading biphenyl and 4-chlorobiphenyl, encode the 2,3-dihydroxybiphenyl 1,2-dioxygenase and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase, respectively. The two genes were sequenced and appear to be present in the order pcbD-pcbC as an operon.

SUBMITTER: Kim E

PROVIDER: S-EPMC167793 | biostudies-other | 1996 Jan

REPOSITORIES: biostudies-other

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Genetic structures of the genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase from biphenyl- and 4-chlorobiphenyl-degrading Pseudomonas sp. strain DJ-12.

Kim E E Kim Y Y Kim C K CK

Applied and environmental microbiology 19960101 1

The pcbC and pcbD genes of Pseudomonas sp. strain DJ-12, a natural isolate degrading biphenyl and 4-chlorobiphenyl, encode the 2,3-dihydroxybiphenyl 1,2-dioxygenase and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase, respectively. The two genes were sequenced and appear to be present in the order pcbD-pcbC as an operon. ...[more]

PMID: 8572703

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Project description: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.

Project description: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.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.

Dataset Information

Genetic structures of the genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase from biphenyl- and 4-chlorobiphenyl-degrading Pseudomonas sp. strain DJ-12.

Publications

Genetic structures of the genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase from biphenyl- and 4-chlorobiphenyl-degrading Pseudomonas sp. strain DJ-12.

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