Characterization of Active Recombinant His-tagged Oxygenase Component of Comamonas testosteroni B-356 Biphenyl Dioxygenase

Hurtubise, Yves; Barriault, Diane; Sylvestre, Michel

doi:10.1074/jbc.271.14.8152

Cited by 56 publications

(76 citation statements)

References 20 publications

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“…The enzyme was purified by affinity chromatography as a His-tagged protein. The UV-visible spectra of the Histagged purified variant BPDOs were identical to that of the parents, exhibiting major peaks at 323 and 455 nm and a shoulder at 575 nm (18). The specific activities toward BPH of the His-tagged purified enzymes for II-11, II-9, and II-10 were 285, 314, and 237 nmol/min/mg, respectively.…”

Section: Resultsmentioning

confidence: 76%

Family Shuffling of a Targeted bphA Region To Engineer Biphenyl Dioxygenase

2002

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In this work we used a new strategy designed to reduce the size of the library that needs to be explored in family shuffling to evolve new biphenyl dioxygenases (BPDOs). Instead of shuffling the whole gene, we have targeted a fragment of bphA that is critical for enzyme specificity. We also describe a new protocol to screen for more potent BPDOs that is based on the detection of catechol metabolites from chlorobiphenyls. Several BphA variants with extended potency to degrade polychlorinated biphenyls (PCBs) were obtained by shuffling critical segments of bphA genes from Burkholderia sp. strain LB400, Comamonas testosteroni B-356, and Rhodococcus globerulus P6. Unlike all parents, these variants exhibited high activity toward 2,2-, 3,3-, and 4,4-dichlorobiphenyls and were able to oxygenate the very persistent 2,6-dichlorobiphenyl. The data showed that the replacement of a short segment ( 335 TFNNIRI 341 ) of LB400 BphA by the corresponding segment ( 333 GINTIRT 339 ) of B-356 BphA or P6 BphA contributes to relax the enzyme toward PCB substrates.Biphenyl dioxygenase (BPDO) is the first enzyme of the biphenyl (BPH) catabolic pathway. BPDO comprises three components: the iron-sulfur oxygenase (ISP BPH ) made up of ␣ (M r ϭ 51,000) and ␤ (M r ϭ 22,000) subunits, the ferredoxin (FER BPH , M r ϭ 12,000), and the ferredoxin reductase (RED BPH , M r ϭ 43,000). The encoding genes for both Burkholderia sp. strain LB400 (15, 16) and Comamonas testosteroni B-356 (35) are bphA (ISP BPH ␣ subunit), bphE (ISP BPH ␤ subunit), bphF (FER BPH ), and bphG (RED BPH ). ISP BPH catalyzes a 2,3-dihydroxylation of BPH. BPDO is of particular interest because of its potential application as biocatalyst to oxygenate priority pollutants such as polychlorinated BPHs (PCBs) or to manufacture fine chemicals.Despite their nearly identical amino acid sequences, the LB400 (15) and Pseudomonas pseudoalcaligenes strain KF707 BPDOs (36) display distinct ranges of PCB substrate (28). LB400 BPDO oxygenates 2,2Ј-dichlorobiphenyl (2,2ЈCB) and exhibits a 3,4-oxygenation of 2,2Ј,5,5ЈCB, whereas KF707 BPDO oxygenates 4,4ЈCB and is poorly active toward the ortho-substituted congeners (28). C. testosteroni B-356 and Rhodococcus globerulus P6 BPDOs, which are more distantly related to KF707 and LB400 BPDOs, poorly catalyze the oxygenation of para-substituted congeners and degrade fairly well 3,3ЈCB (11, 19). There is 75% amino acid sequence identity between the B-356 and LB400 BphAs (35), 64% amino acid sequence identity between P6 BphA1 and LB400 BphA (2), and 65% amino acid sequence identity between P6 BphA1 and B-356 BphA (35).Variant BPDOs that had inherited the catalytic features of both parents and exhibiting extended activities toward several tetra-and penta-substituted congeners were obtained by shuffling the very closely related LB400 bphA with KF707 bphA1 (9, 23). However, no novel BPDO has yet been described which can efficiently catalyze oxygenation of the most persistent congeners.Family shuffling of genes of lesser homology increases the sequenc...

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Section: Resultsmentioning

confidence: 76%

Family Shuffling of a Targeted bphA Region To Engineer Biphenyl Dioxygenase

2002

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“…His-tagged purified enzyme components were produced and purified by following protocols published previously (31). The enzyme assays were performed in a volume of 200 l in 50 mM morpholineethanesulfonic (MES) buffer, pH 6.0, at 37°C as described previously (32). For metabolite analyses, the reaction medium was incubated for 10 min and the metabolites were extracted at pH 6.0 with ethyl acetate, and then they were treated with nBuB or TMS for GC-MS analyses as described above.…”

Section: Methodsmentioning

confidence: 99%

Has the Bacterial Biphenyl Catabolic Pathway Evolved Primarily To Degrade Biphenyl? The Diphenylmethane Case

Pham

Sylvestre

2013

J Bacteriol

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In this work, we have compared the ability of Pandoraea pnomenusa B356 and of Burkholderia xenovorans LB400 to metabolize diphenylmethane and benzophenone, two biphenyl analogs in which the phenyl rings are bonded to a single carbon. Both chemicals are of environmental concern. P. pnomenusa B356 grew well on diphenylmethane. On the basis of growth kinetics analyses, diphenylmethane and biphenyl were shown to induce the same catabolic pathway. The profile of metabolites produced during growth of strain B356 on diphenylmethane was the same as the one produced by isolated enzymes of the biphenyl catabolic pathway acting individually or in coupled reactions. The biphenyl dioxygenase oxidizes diphenylmethane to 3-benzylcyclohexa-3,5-diene-1,2-diol very efficiently, and ultimately this metabolite is transformed to phenylacetic acid, which is further metabolized by a lower pathway. Strain B356 was also able to cometabolize benzophenone through its biphenyl pathway, although in this case, this substrate was unable to induce the biphenyl catabolic pathway and the degradation was incomplete, with accumulation of 2-hydroxy-6,7-dioxo-7-phenylheptanoic acid. Unlike strain B356, B. xenovorans LB400 did not grow on diphenylmethane. Its biphenyl pathway enzymes metabolized diphenylmethane, but they poorly metabolize benzophenone. The fact that the biphenyl catabolic pathway of strain B356 metabolized diphenylmethane and benzophenone more efficiently than that of strain LB400 brings us to postulate that in strain B356, this pathway evolved divergently to serve other functions not related to biphenyl degradation.

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“…In some experiments, the metabolites were analyzed from catalytic oxygenation using reconstituted BPDO prepared from His-tagged purified enzymes components that were obtained following protocols published previously (2). Catalytic activities were determined from measurement of substrate depletion recorded by GC-MS analysis (15).…”

Section: Whole Cell Assays To Evaluate the Pcb Degrading Potency-e Colimentioning

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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Characterization of Active Recombinant His-tagged Oxygenase Component of Comamonas testosteroni B-356 Biphenyl Dioxygenase

Cited by 56 publications

References 20 publications

Family Shuffling of a Targeted bphA Region To Engineer Biphenyl Dioxygenase

Family Shuffling of a Targeted bphA Region To Engineer Biphenyl Dioxygenase

Has the Bacterial Biphenyl Catabolic Pathway Evolved Primarily To Degrade Biphenyl? The Diphenylmethane Case

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

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