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

Barriault, Diane; Sylvestre, Michel

doi:10.1074/jbc.m406805200

Cited by 48 publications

(62 citation statements)

References 29 publications

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“…Variant p4 is an evolved BPDO obtained by simultaneous random mutagenesis of the amino acid residues of region III of LB400 BphA (30). The sequence pattern of p4 BphA is identical to that of LB400 BphA except for the mutations T335A and F336M.…”

Section: Methodsmentioning

confidence: 99%

“…The sequence pattern of p4 BphA is identical to that of LB400 BphA except for the mutations T335A and F336M. Plasmids pQE31[LB400-bphAE], pQE31[B-356-bphAE], and pQE31[p4-bphAE] were described previously (16,30). The E. coli strains bearing these plasmids expressed His-tagged ISP BPH (16).…”

Section: Methodsmentioning

confidence: 99%

“…Several reports (11,14,16,30) show that the sequence pattern of a stretch of seven amino acids of the C-terminal portion of BphA called region III strongly influences the range of PCB congeners that the enzyme can oxygenate efficiently, as well as its regiospecificity toward 2,2Ј-CB. Other amino acids, including residue 377 of LB400 BphA (11,16) and several other residues that, according to a model of BPDO, have apparently no contact with the substrate (16), seem to act in association with residues of region III to influence the reaction turnover rates and regiospecificity toward chlorobiphenyls.…”

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

confidence: 99%

“…It also exhibited an altered regiospecificity toward 2,2Ј-CB, producing as a major metabolite the dihydro-dihydroxy-dichlorobiphenyl normally reported as a minor metabolite by LB400 BPDO. However, the fact that the dihydroxy chlorobiphenyl derived from this metabolite was not cleaved by BphC (30) suggested that the oxygenation occurred on the meta and para carbons 3 and 4 or 4 and 5 of 2,2Ј-CB rather than on the ortho and meta carbons 5 and 6. This assumption was confirmed in the present study.…”

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

confidence: 99%

“…This variant catalyzes the oxygenation of many PCB congeners more efficiently than LB400 BPDO (30). It also exhibited an altered regiospecificity toward 2,2Ј-CB, producing as a major metabolite the dihydro-dihydroxy-dichlorobiphenyl normally reported as a minor metabolite by LB400 BPDO.…”

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

confidence: 99%

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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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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

confidence: 99%

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

confidence: 99%

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

confidence: 99%

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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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Biological and Biomedical Aspects of Metal Phenolates

Ming

2014

Patai's Chemistry of Functional Groups

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The phenol functionality is involved in diverse biological processes and functions, serving as a proton donor and as a metal‐binding ligand. It is commonly found in many biochemicals (such as siderophores and the amino acids tyrosine and tryptophan), pharmaceuticals (including antibiotics, metal‐chelating agents, and diagnostic agents), and natural products (such as polyphenols, flavonoids, and salicylic acid). In association with other functional groups, the phenol moiety forms various types of metal‐binding sites of diverse structures and functions, for example, for iron binding and transport, in enzymes for oxygenation of substrates, and for biotransformation of aromatic compounds. Many environmentally hazardous aromatic compounds such as bisphenol A, PCB, and DDT can be degraded by tyrosinate(phenolate)‐containing metalloenzymes from microorganisms, which provides a clue for bioremediation of these toxic substances. Moreover, the phenol‐containing wet‐adhesive byssal proteins from some clams and mussels has stimulated further development of adhesive materials for medical and other applications; and the study of binding of transferrin with nanoparticles affords better understanding of protein–mineral interfacial interactions. In addition to these naturally occurring metal‐phenolate centers, there are many metal complexes of phenol and derivatives that exhibit various catalytic activities and serve as structural and/or functional model systems for tyrosinate‐containing metalloproteins.

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Application of Aromatic Hydrocarbon Dioxygenases

Tan

Parales

2016

Green Biocatalysis

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c h a p t e r 1 7 INTRODUCTIONThe first reported aromatic hydrocarbon dioxygenase, toluene dioxygenase, was identified to catalyze cis-dihydroxylation of benzene and toluene in Pseudomonas putida F1 [1, 2]. Since then, many more dioxygenases have been studied, and the crys tal structures of several have been determined [3]. This group of enzymes plays an important role in the initial step in the biodegradation of both naturally occurring and man-made aromatic compounds, including aromatic acids, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and nitroaromatic, aminoaromatic and halogenated aromatic compounds [4,5]. Dioxygenases are nonheme Rieske-type NAD(P)H-dependent enzymes that intro duce both atoms of molecular oxygen into their substrates. The multicomponent enzyme systems are composed of a catalytic oxygenase component and one-or two-electron trans fer proteins, including a flavoprotein reductase, and in some cases a ferredoxin that medi ates electron transfer from the reductase to the oxygenase component ([3]; Figure 17.1).In general, Rieske dioxygenases are capable of oxidizing a broad range of substrates, well beyond the range of compounds that serves as growth substrates for the host bacterium [4,5,7]. For example, toluene dioxygenase from P. putida F1 facilitates the oxidation of over 200 different substrates [7], and naphthalene dioxygenase from Pseudomonas sp. NCIB 9816-4 has been documented to oxidize over 60 different substrates [8]. In addition, these enzymes catalyze diverse types of reactions, including cisdihydroxylations, O-and N-dealkylations, desaturations, and the formation of chiral sulfoxides from sulfides ([7-9]; Figure 17.2). In many cases the products are chiral com pounds that are high in enantiomeric purity [8,10,11]. For the aforementioned reasons, dioxygenases are attractive biocatalysts for bioremediation and industrial applications. CHALLENGES IN AROMATIC HYDROCARBON DIOXYGENASE APPLICATIONSThe practical use of dioxygenases in industry is generally limited to the use of whole-cell biocatalysts due to several challenges. The catalytic activity of dioxygenases is dependent on reducing equivalents provided by NADH or NADPH, which require regeneration by electron transfer proteins [12,13]. The regeneration process is typically a limiting step

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

Cited by 48 publications

References 29 publications

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

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

Biological and Biomedical Aspects of Metal Phenolates

Application of Aromatic Hydrocarbon Dioxygenases

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