Characterization of hybrid biphenyl dioxygenases obtained by recombiningBurkholderiasp. strain LB400bphAwith the homologous gene ofComamonas testosteroniB-356

Barriault, Diane; Simard, C; Chatel, Henrietta; Sylvestre, Michel

doi:10.1139/w01-108

Cited by 16 publications

(11 citation statements)

References 25 publications

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“…Plasmid pQE31[LB-400-bphAE], in which bphA was mutated to introduce an AvrII site at position 1354, was described previously (14); it encodes for a fused His-tagged ISP BPH (14,15) carrying the His tag on BphA. Plasmid pYH31[LB400-bphF-GBC] was described previously (16).…”

Section: Methodsmentioning

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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Section: Methodsmentioning

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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“…Studies on various biphenyl 2,3-dioxygenases have revealed considerable differences in their congener selectivity patterns, as well as their preference for the attacked ring (4,12,22,28,38,44).…”

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confidence: 99%

Substrate Specificity and Expression of Three 2,3-Dihydroxybiphenyl 1,2-Dioxygenases from Rhodococcus globerulus Strain P6

et al. 2003

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Rhodococcus globerulus strain P6 contains at least three genes, bphC1, bphC2, and bphC3, coding for 2,3-dihydroxybiphenyl 1,2-dioxygenases; the latter two specify enzymes of the family of one-domain extradiol dioxygenases. In order to assess the importance of these different isoenzymes for the broad catabolic activity of this organism towards the degradation of polychlorinated biphenyls (PCBs), the capacities of recombinant enzymes expressed in Escherichia coli to transform different chlorosubstituted dihydroxybiphenyls formed by the action of R. globerulus P6 biphenyl dioxygenase and biphenyl 2,3-dihydrodiol dehydrogenase were determined. Whereas both BphC2 and BphC3 showed similar activities for 2,3-dihydroxybiphenyl and all monochlorinated 2,3-dihydroxybiphenyls, BphC1 exhibited only weak activity for 2-chloro-2,3-dihydroxybiphenyl. More highly chlorinated 2-chlorosubstituted 2,3-dihydroxybiphenyls were also transformed at high rates by BphC2 and BphC3 but not BphC1. In R. globerulus P6, BphC2 was constitutively expressed, BphC1 expression was induced during growth on biphenyl, and BphC3 was not expressed at significant levels under the experimental conditions. Although we cannot rule out the expression of BphC3 under certain environmental conditions, it seems that the contrasting substrate specificities of BphC1 and BphC2 contribute significantly to the versatile PCB-degrading phenotype of R. globerulus P6.

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“…We note that these findings contrast those of Hurtubise et al (1998) and Chebrou et al (1999), who reported a significant contribution of the β subunit to BDO-substrate interaction. However, a recent publication from this group (Barriault et al, 2001) reported no major influence of the small subunit on substrate specificity. Based on all fusions generated, the α subunit may be divided into eight segments, designated A to H (Fig.…”

Section: Discussionmentioning

confidence: 91%

“…Although investigations of different ARHDOs are of limited comparability, we note that Beil et al (1998) and Parales et al (2000) reported effects of amino acid substitutions at five or two positions, respectively, within the corresponding regions of a toluene or a naphthalene dioxygenase, respectively. Barriault et al (2001), however, exchanging a region between BDOs of strains LB400 and B-356 that comprises the region substituted in BDO-H30, found only a minor influence on substrate specificity. These differences in results may be a consequence of the different assays used.…”

Section: Discussionmentioning

confidence: 91%

“…2) that harbours the active site. Barriault et al (2001) exchanged the αA regions between the BDOs from Comamonas testosteroni B-356 and strain LB400, which differ at 28 positions. Whilst the B-356 variant containing αA from strain LB400 behaved very similarly to the B-356 WT, the reverse replacement showed significant differences in turnover of two of the four CBs assayed.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

The principal determinants for the structure of the substrate-binding pocket are located within a central core of a biphenyl dioxygenase α subunit

2002

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Protein engineering by segment exchange was used to distinguish between regions of major and minor influence on the structure of the substrate-binding pocket of a biphenyl dioxygenase (BDO). Eight chimaeric enzyme systems were generated that each consisted of a hybrid hydroxylase α subunit (BphA1) containing segments from Burkholderia sp. strain LB400 and Rhodococcus globerulus P6, and of a hydroxylase β subunit (BphA2), a ferredoxin (BphA3) and a ferredoxin reductase (BphA4) from strain LB400. All hybrid bphA1 genes were expressed at high levels. Seven of the resulting fusion subunits functionally interacted with the other polypeptides of the dioxygenase system to yield catalytically active enzymes. Changes in the regiospecificity of substrate attack, monitored by the formation of seventeen different dioxygenation products obtained from seven chlorobiphenyls, were used to monitor effects of segment exchanges on the structure of the BDO substratebinding site. Exchanges of neither the β subunit nor the N-and C-terminal regions of the α subunit exerted significant influences. All BDO regions that showed major effects on the substrate-binding pocket were located between approximately positions 165 and 395 of the α subunit. Within this part of the enzyme, in addition to segments identified previously, a subregion which is involved in ligation of the mononuclear iron significantly influenced the regiospecificity of substrate dioxygenation. Moreover, the results indicate that the construction of appropriate hybrid genes may be used as a general strategy to overcome problems in obtaining heterologous BDO activities in Escherichia coli or other host organisms.

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Characterization of hybrid biphenyl dioxygenases obtained by recombiningBurkholderiasp. strain LB400bphAwith the homologous gene ofComamonas testosteroniB-356

Cited by 16 publications

References 25 publications

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

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

Substrate Specificity and Expression of Three 2,3-Dihydroxybiphenyl 1,2-Dioxygenases from Rhodococcus globerulus Strain P6

The principal determinants for the structure of the substrate-binding pocket are located within a central core of a biphenyl dioxygenase α subunit

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