Sequencing of Comamonas testosteroni strain B-356-biphenyl/chlorobiphenyl dioxygenase genes: evolutionary relationships among Gram-negative bacterial biphenyl dioxygenases

Sylvestre, Michel; Suquet, Marc; Hurtubise, Yves; Bergeron, Jean‐Marie; Ahmad, Darakhshan; Shareck, François; Barriault, Diane; Guillemette, Isabelle; Juteau, Jean Marc

doi:10.1016/0378-1119(96)00039-x

Cited by 64 publications

(43 citation statements)

References 29 publications

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“…As a consequence, the bph genes are present on bacterial chromosomes (2,3,11,20,29,35,39,54,78), plasmids (30,65,85), and transposons (45,51,59,73). The chromosomal 90-kb element (termed the bph-sal element) containing a bph gene cluster in Pseudomonas putida KF715 can be transferred to other P. putida strains at a high frequency (59).…”

Section: Structural Versatilities Of Biphenyl Catabolic Bph Genesmentioning

confidence: 99%

Biphenyl Dioxygenases: Functional Versatilities and Directed Evolution

2004

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Section: Structural Versatilities Of Biphenyl Catabolic Bph Genesmentioning

confidence: 99%

Biphenyl Dioxygenases: Functional Versatilities and Directed Evolution

2004

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“…The second and third components are a flavoprotein reductase and a ferredoxin that are involved in the transfer of electrons from NADH to ISP BPH . BPDO components are coded by bphA (␣-subunit of ISP BPH ), bphE (␤-subunit of ISP BPH ), bphF (ferredoxin) and bphG (flavoprotein reductase) in Burkholderia xenovorans LB400 (7) (also called Burkholderia fungorum LB400 or Pseudomonas cepacia LB400) (8) and in Comamonas testosteroni B-356 (9).…”

Section: (2 3) the Cis-(2r3s)-dihydroxy-1-phenylcyclohexa-46-dienmentioning

confidence: 99%

Revisiting the Regiospecificity of Burkholderia xenovorans LB400 Biphenyl Dioxygenase toward 2,2′-Dichlorobiphenyl and 2,3,2′,3′-Tetrachlorobiphenyl

Barriault

Lépine

Mohammadi

et al. 2004

Journal of Biological Chemistry

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2,2 -Dichlorobiphenyl (CB) is transformed by the biphenyl dioxygenase of Burkholderia xenovorans LB400 (LB400 BPDO) into two metabolites (1 and 2). The most abundant metabolite, 1, was previously identified as 2,3-dihydroxy-2 -chlorobiphenyl and was presumed to originate from the initial attack by the oxygenase on the chlorine-bearing ortho carbon and on its adjacent meta carbon of one phenyl ring. 2,3,2 ,3 -Tetrachlorobiphenyl is transformed by LB400 BPDO into two metabolites that had never been fully characterized structurally. We determined the precise identity of the metabolites produced by LB400 BPDO from 2,2 -CB and 2,3,2 ,3 -CB, thus providing new insights on the mechanism by which 2,2 -CB is dehalogenated to generate 2,3-dihydroxy-2 -chlorobiphenyl. We reacted 2,2 -CB with the BPDO variant p4, which produces a larger proportion of metabolite 2. The structure of this compound was determined as cis-3,4-dihydro-3,4-dihydroxy-2,2 -dichlorobiphenyl by NMR. Metabolite 1 obtained from 2,2 -CB-d 8 was determined to be a dihydroxychlorobiphenyl-d 7 by gas chromatographic-mass spectrometric analysis, and the observed loss of only one deuterium clearly shows that the oxygenase attack occurs on carbons 2 and 3. An alternative attack at the 5 and 6 carbons followed by a rearrangement leading to the loss of the ortho chlorine would have caused the loss of more than one deuterium. The major metabolite produced from catalytic oxygenation of 2,3,2 ,3 -CB by LB400 BPDO was identified by NMR as cis-4,5-dihydro-4,5-dihydroxy-2,3,2 ,3 -tetrachlorobiphenyl. These findings show that LB400 BPDO oxygenates 2,2 -CB principally on carbons 2 and 3 and that BPDO regiospecificity toward 2,2 -CB and 2,3,2, ,3 -CB disfavors the dioxygenation of the chlorine-free orthometa carbons 5 and 6 for both congeners.The enzymes of the bacterial biphenyl catabolic pathway are very versatile. They can co-metabolically transform several polychlorinated biphenyls (1). The initial reaction of this pathway is catalyzed by the biphenyl dioxygenase (BPDO) 1 (2, 3). The cis- (2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene (commonly called cis-2,3-dihydro-2,3-dihydroxybiphenyl) generated by the catalytic oxygenation of biphenyl is dehydrogenated by the 2,3-dihydro-2,3-dihydroxybiphenyl-2,3-dehydrogenase (BphB) and the catechol produced is cleaved by the 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC). The 2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoic acid produced is then hydrolyzed by the 2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoic acid hydrolase to yield benzoate and 2-hydroxypentanoate (Fig. 1). The substrate specificity of BPDO is crucial, because it limits the range of compounds that can potentially be degraded by the catabolic system. BPDO is a three-component enzymatic system (4 -6) (Fig. 1). The first component is an iron-sulfur protein (ISP BPH ) that interacts with the substrate to catalyze the addition of molecular oxygen. The second and third components are a flavoprotein reductase and a ferredoxin that are involved in the transfer of electrons from N...

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“…A stretch of seven amino acid residues termed region III was of particular interest (6,9). The sequence of amino acids for region III of B. xenovorans LB400 BPDO, which is the wild-type enzyme exhibiting the highest PCB degrading potency, is Thr 335 -Phe 336 -Asn 337 -Asn 338 -Ile 339 -Arg 340 -Ile 341 (4 (11). KF707 BphA BPDO shares more than 95% homology with LB400 BphA (9).…”

mentioning

confidence: 99%

Evolution of the Biphenyl Dioxygenase BphA from Burkholderia xenovorans LB400 by Random Mutagenesis of Multiple Sites in Region III

Barriault

Sylvestre

2004

Journal of Biological Chemistry

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It is now established that several amino acids of region III of the biphenyl dioxygenase (BPDO) ␣ subunit are involved in substrate recognition and regiospecificity toward chlorobiphenyls. However, the sequence pattern of the amino acids of that segment of seven amino acids located in the C-terminal portion of the ␣ subunit is rather limited in BPDOs of natural occurrence. In this work, we have randomly mutated simultaneously four residues (Thr 335 -Phe 336 -Ile 338 -Ile 341 ) of region III of Burkholderia xenovorans LB400 BphA. The library was screened for variants able to oxygenate 2,2 -dichlorobiphenyl (2,2 -CB). Replacement of Phe 336 with Met or Ile with a concomitant change of Thr 335 to Ala created new variants that transformed 2,2 -CB into 3,4-dihydro-3,4-dihydroxy-2,2 -dichlorobiphenyl, which is a dead end metabolite that was not cleaved by BphC. Replacement of Thr 335 -Phe 336 with Ala 335 -Leu 336 did not cause this type of phenotypic change. Regiospecificity toward congeners other than 2,2 -CB that were oxygenated more efficiently by variant Ala 335 -Met 336 than by LB400 BPDO was similar for both enzymes. Thus structural changes that altered the regiospecificity toward 2,2 -CB did not affect the metabolite profile of other congeners, although it affected the rate of conversion of these congeners. It was especially noteworthy that both LB400 BPDO and the Ala 335 -Met 336 variant generated 2,3-dihydroxy-2 ,4,4 -trichlorobiphenyl as the sole metabolite from 2,4,2 ,4 -CB and 4,5-dihydro-4,5-dihydroxy-2,3,2 ,3 -tetrachlorobiphenyl as the major metabolite from 2,3,2 ,3 -CB. This shows that 2,4,2 ,4 -CB is oxygenated principally onto vicinal ortho-meta carbons 2 and 3 and that 2,3,2 ,3 -CB is oxygenated onto meta-para carbons 4 and 5 by both enzymes. The data suggest that interactions between the chlorine substitutes on the phenyl ring and specific amino acid residues of the protein influence the orientation of the phenyl ring inside the catalytic pocket.Biphenyl dioxygenase (BPDO) 1 catalyzes the first reaction of the biphenyl catabolic pathway. BPDO comprises three components (1, 2): The iron-sulfur oxygenase (ISP BPH ) made up of an ␣ subunit (M r ϭ 51,000) and a ␤ subunit (M r ϭ 22,000), the ferredoxin (FER BPH , M r ϭ 12,000), and the ferredoxin reductase (RED BPH , M r ϭ 43,000). The encoding genes for Burkholderia xenovorans LB400 (3), which is also called Burkholderia (Pseudomonas) sp. LB400 (4, 5), are bphA (ISP BPH ␣ subunit), bphE (ISP BPH ␤ subunit), bphF (FER BPH ), and bphG (RED BPH ). BPDO can oxygenate several polychlorinated biphenyl (PCB) congeners. The application of BPDO for efficient PCB degrading processes will require an expansion of the range of PCB substrates it can oxygenate. The catalytic oxygenation of biphenyl occurs normally on carbons 2 and 3 to generate the cis-2,3-dihydro-2,3-dihydroxybiphenyl, which is dehydrogenated by the cis-2,3-dihydro-2,3-dihydroxybiphenyl 2,3-dehydrogenase encoded by bphB in strain LB400. The resulting catechol 2,3-dihydroxybiphenyl is then cleaved b...

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Sequencing of Comamonas testosteroni strain B-356-biphenyl/chlorobiphenyl dioxygenase genes: evolutionary relationships among Gram-negative bacterial biphenyl dioxygenases

Cited by 64 publications

References 29 publications

Biphenyl Dioxygenases: Functional Versatilities and Directed Evolution

Biphenyl Dioxygenases: Functional Versatilities and Directed Evolution

Revisiting the Regiospecificity of Burkholderia xenovorans LB400 Biphenyl Dioxygenase toward 2,2′-Dichlorobiphenyl and 2,3,2′,3′-Tetrachlorobiphenyl

Evolution of the Biphenyl Dioxygenase BphA from Burkholderia xenovorans LB400 by Random Mutagenesis of Multiple Sites in Region III

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