Dehalogenation, Denitration, Dehydroxylation, and Angular Attack on Substituted Biphenyls and Related Compounds by a Biphenyl Dioxygenase

Seeger, Michael; Cámara, Beatríz; Höfer, Bernd

doi:10.1128/jb.183.12.3548-3555.2001

Cited by 96 publications

(60 citation statements)

References 41 publications

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“…Helium served as carrier gas. The mass spectrometer was operated in the electron impact ionization mode at 70 eV as described (Seeger et al, 2001(Seeger et al, , 2003. The following AHL standards were purchased from Fluka: N-hexanoyl-DL-homoserine lactone (AHL-C 6 ), N-octanoyl-DLhomoserine lactone (AHL-C 8 ) and Ntetradecanoyl-DL-homoserine lactone (AHL-C 14 ).…”

Section: Purification and Identification Of Ahlmentioning

confidence: 99%

A Lux-like Quorum Sensing System in the Extreme Acidophile Acidithiobacillus ferrooxidans

Rivas

Seeger²,

Holmes

et al. 2005

Biol. Res.

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The genome of the acidophilic, proteobacterium Acidithiobacillus ferrooxidans, contains linked but divergently oriented genes, termed afeI and afeR, whose predicted protein products are significantly similar to the LuxI and LuxR families of proteins. A possible promoter and Lux box are predicted upstream of afeI. A cloned copy of afeI, expressed in E. coli, encodes an enzyme that catalyzes the production of a diffusible compound identified by gas chromatography and mass spectrometry as an unsubstituted N-acyl homoserine lactone (AHL) of chain length C 14 . This AHL can be detected by a reporter strain of Sinorhizobium meliloti Rm41 suggesting that it is biologically active. The reporter strain also responds to extracts of the supernatant of A. ferrooxidans grown to early stationary phase in sulfur medium indicating that a diffusible AHL is produced by this microorganism. Semi-quantitative RT-PCR experiments indicate that afeI and afeR are expressed maximally in early stationary phase and are more expressed when A. ferrooxidans is grown in sulfur-rather than iron-containing medium. Given the predicted amino acid sequence and functional properties of AfeI and AfeR it is proposed that A. ferrooxidans has a quorum sensing system similar to the LuxI-LuxR paradigm.

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Section: Purification and Identification Of Ahlmentioning

confidence: 99%

A Lux-like Quorum Sensing System in the Extreme Acidophile Acidithiobacillus ferrooxidans

Rivas

Seeger²,

Holmes

et al. 2005

Biol. Res.

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“…The latter were presumed to generate a metabolite that BphC was unable to cleave. Therefore, these variants were expected to have changed their regiospecificity toward 2,2Ј-CB because it has been established that catalytic oxygenation of this congener by LB400 BPDO produces as major metabolite 2,3-dihydroxy-2Ј-chlorobiphenyl (20,21) that can be cleaved by BphC. These pinkish gray variants were also characterized.…”

Section: Screening For Bpdo Variants Able To Oxygenate 22ј-cb-tomentioning

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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“…The second metabolite obtained from 2,2Ј-CB (representing 10% or less of the product) was presumed to be the 5,6-dihydro-5,6-dihydroxy-2,2Ј-dichlorobiphenyl (2) based on the hypothesis that the enzyme normally catalyzes the oxygenation of vicinal ortho and meta carbons of biphenyl. Several other reports used these assumptions to interpret the metabolism of 2,2Ј-CB by engineered enzymes (13)(14)(15). In fact, in recent reports (13)(14)(15), most of the conclusions drawn about regiospecificity alterations toward 2,2Ј-CB that occurred after the engineering of BPDOs were based on assumptions that need confirmation.…”

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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Dehalogenation, Denitration, Dehydroxylation, and Angular Attack on Substituted Biphenyls and Related Compounds by a Biphenyl Dioxygenase

Cited by 96 publications

References 41 publications

A Lux-like Quorum Sensing System in the Extreme Acidophile Acidithiobacillus ferrooxidans

A Lux-like Quorum Sensing System in the Extreme Acidophile Acidithiobacillus ferrooxidans

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

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

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