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

It is now established that several amino acids of region III of the biphenyl dioxygenase (BPDO) α 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 α subunit is rather limited in BPDOs of natural occurrence. In this work, we have randomly mutated simultaneously four residues (Thr335-Phe336-Ile338-Ile341) 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 Phe336 with Met or Ile with a concomitant change of Thr335 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 Thr335-Phe336 with Ala335-Leu336 did not cause this type of phenotypic change. Regiospecificity toward congeners other than 2,2′-CB that were oxygenated more efficiently by variant Ala335-Met336 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 Ala335-Met336 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. It is now established that several amino acids of region III of the biphenyl dioxygenase (BPDO) α 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 α subunit is rather limited in BPDOs of natural occurrence. In this work, we have randomly mutated simultaneously four residues (Thr335-Phe336-Ile338-Ile341) 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 Phe336 with Met or Ile with a concomitant change of Thr335 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 Thr335-Phe336 with Ala335-Leu336 did not cause this type of phenotypic change. Regiospecificity toward congeners other than 2,2′-CB that were oxygenated more efficiently by variant Ala335-Met336 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 Ala335-Met336 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) 1The abbreviations used are: BPDO, biphenyl dioxygenase; PCB, polychlorinated biphenyl; 4-CB, 4-chlorobiphenyl; 2,2′-CB, 2,2′-dichlorobiphenyl; 3,3′-CB, 3,3′-dichlorobiphenyl; 4,4′-CB, 4,4′-dichlorobiphenyl; 2,6-CB, 2,6-dichlorobiphenyl; 2,4,3′-CB, 2,4,3′-trichlorobiphenyl; 2,4,4′-CB, 2,4,4′-trichlorobiphenyl; 2,3,4′-CB, 2,3,4′-trichlorobiphenyl; 2,6,2′,6′-CB, 2,6,2′,6′-tetrachlorobiphenyl; 2,5,2′,5′-CB, 2,5,2′,5′-tetrachlorobiphenyl; 2,3,2′,3′-CB, 2,3,2′3′-tetrachlorobiphenyl; 2,4,2′,4′-CB, 2,4,2′,4′-tetrachlorobiphenyl; 2,4,3′,4′-CB, 2,4,3′,4′-tetrachlorobiphenyl; 3,4,3′,4′-CB, 3,4,3,4′-tetrachlorobiphenyl; 3,4,3′,5′-CB, 3,4,3′5′-tetrachlorobiphenyl; 2,3,4,2′,5′-CB, 2,3,4,2′,5′-pentachlorobiphenyl; 2,4,5,3′,4′-CB, 2,4,5,3′,4′-pentachlorobiphenyl; 2,4,5,2′,4′,5′-CB, 2,4,5,2′,4′,5′-hexachlorobiphenyl; 2,3,4,5,2′,5′-CB, 2,3,4,5,2′,5′-hexachlorobiphenyl; HOPDA, 2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoic acid; IPTG, isopropyl β-d-thiogalactopyranoside; GC-MS, gas chromatography-mass spectrometry.1The abbreviations used are: BPDO, biphenyl dioxygenase; PCB, polychlorinated biphenyl; 4-CB, 4-chlorobiphenyl; 2,2′-CB, 2,2′-dichlorobiphenyl; 3,3′-CB, 3,3′-dichlorobiphenyl; 4,4′-CB, 4,4′-dichlorobiphenyl; 2,6-CB, 2,6-dichlorobiphenyl; 2,4,3′-CB, 2,4,3′-trichlorobiphenyl; 2,4,4′-CB, 2,4,4′-trichlorobiphenyl; 2,3,4′-CB, 2,3,4′-trichlorobiphenyl; 2,6,2′,6′-CB, 2,6,2′,6′-tetrachlorobiphenyl; 2,5,2′,5′-CB, 2,5,2′,5′-tetrachlorobiphenyl; 2,3,2′,3′-CB, 2,3,2′3′-tetrachlorobiphenyl; 2,4,2′,4′-CB, 2,4,2′,4′-tetrachlorobiphenyl; 2,4,3′,4′-CB, 2,4,3′,4′-tetrachlorobiphenyl; 3,4,3′,4′-CB, 3,4,3,4′-tetrachlorobiphenyl; 3,4,3′,5′-CB, 3,4,3′5′-tetrachlorobiphenyl; 2,3,4,2′,5′-CB, 2,3,4,2′,5′-pentachlorobiphenyl; 2,4,5,3′,4′-CB, 2,4,5,3′,4′-pentachlorobiphenyl; 2,4,5,2′,4′,5′-CB, 2,4,5,2′,4′,5′-hexachlorobiphenyl; 2,3,4,5,2′,5′-CB, 2,3,4,5,2′,5′-hexachlorobiphenyl; HOPDA, 2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoic acid; IPTG, isopropyl β-d-thiogalactopyranoside; GC-MS, gas chromatography-mass spectrometry. catalyzes the first reaction of the biphenyl catabolic pathway. BPDO comprises three components (1Haddock J.D. Gibson D.T. J. Bacteriol. 1995; 177: 5834-5839Crossref PubMed Google Scholar, 2Hurtubise Y. Barriault D. Sylvestre M. J. Biol. Chem. 1996; 271: 8152-8156Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar): The iron-sulfur oxygenase (ISPBPH) made up of an α subunit (Mr = 51,000) and a β subunit (Mr = 22,000), the ferredoxin (FERBPH, Mr = 12,000), and the ferredoxin reductase (REDBPH, Mr = 43,000). The encoding genes for Burkholderia xenovorans LB400 (3Denef V.J. Park J. Tsoi T.V. Rouillard J.M. Zhang H. Wibbenmeyer J.A. Verstraete W. Gulari E. Hashsham S.A. Tiedje J.M. Appl. Environ. Microbiol. 2004; 70: 4961-4970Crossref PubMed Scopus (95) Google Scholar), which is also called Burkholderia (Pseudomonas) sp. LB400 (4Erickson B.D. Mondello F.J. J. Bacteriol. 1992; 174: 2903-2912Crossref PubMed Google Scholar, 5Fain M.G. Haddock J.D. Curr. Microbiol. 2001; 42: 269-275PubMed Google Scholar), are bphA (ISPBPH α subunit), bphE (ISPBPH β subunit), bphF (FERBPH), and bphG (REDBPH). 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 by the 2,3-dihydroxybiphenyl 1,2-dioxygenase encoded by bphC in strain LB400. 2-Hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoic acid (HOPDA) is then hydrolyzed by the HOPDA hydrolase (BphD) to yield benzoate and 2-hydroxypentanoic acid (Fig. 1). Based on site-directed mutagenesis and family shuffling of genes, variant BPDOs with extended PCB degrading potency were obtained (6Barriault D. Plante M.M. Sylvestre M. J. Bacteriol. 2002; 184: PubMed Scopus Google Scholar, W. PubMed Scopus Google Scholar, H. M. PubMed Scopus Google Scholar, F.J. B.D. Appl. Environ. Microbiol. PubMed Google However, several congeners that are of for are not by the BPDO variants to acid residues of the C-terminal portion of the α subunit were to influence the substrate of the (6Barriault D. Plante M.M. Sylvestre M. J. Bacteriol. 2002; 184: PubMed Scopus Google Scholar, W. PubMed Scopus Google Scholar, H. M. PubMed Scopus Google Scholar, F.J. B.D. Appl. Environ. Microbiol. PubMed Google of seven amino acid residues region III was of (6Barriault D. Plante M.M. Sylvestre M. J. Bacteriol. 2002; 184: PubMed Scopus Google Scholar, F.J. B.D. Appl. Environ. Microbiol. PubMed Google The sequence of amino acids for region III of xenovorans LB400 BPDO, which is the the PCB degrading is (4Erickson B.D. Mondello F.J. J. Bacteriol. 1992; 174: 2903-2912Crossref PubMed Google The sequence pattern of region is in J. J. Biol. Chem. 1992; Full Text PDF PubMed Google and in strain M. M. Y. J. D. Barriault D. J.M. 1996; 174: PubMed Scopus Google BPDO more than with LB400 F.J. B.D. Appl. Environ. Microbiol. PubMed Google BPDO and BPDO the oxygenation of a range of PCB congeners than LB400 of region III of LB400 by that of or of created that oxygenated a range of PCB congeners (6Barriault D. Plante M.M. Sylvestre M. J. Bacteriol. 2002; 184: PubMed Scopus Google Scholar, H. M. PubMed Scopus Google Scholar, F.J. B.D. Appl. Environ. Microbiol. PubMed Google Scholar, H. M. J. Bacteriol. 2002; 184: PubMed Scopus Google This shows that region III amino acids are not the the catalytic toward it also shows that the sequence pattern of region III of LB400 is not for catalytic toward chlorobiphenyls. In to influence on the range of used as substrate by the amino acids of region III also to in regiospecificity toward Thus H. M. J. Bacteriol. 2002; 184: PubMed Scopus Google that of with as in LB400 the regiospecificity toward 2,2′-dichlorobiphenyl to of The PCB congeners as and are the to LB400 BPDO is the BPDO of natural that can oxygenate 2,2′-CB efficiently F.J. B.D. Appl. Environ. Microbiol. PubMed Google In we that BPDOs for to oxygenate 2,2′-CB a rate than the LB400 BPDO were able to a range of PCB congeners than LB400 BPDO (6Barriault D. Plante M.M. Sylvestre M. J. Bacteriol. 2002; 184: PubMed Scopus Google the amino acid residues of region III influence the range of PCB congeners oxygenated by the on the of of that are in data the of sequence of this of amino acids is rather limited (6Barriault D. Plante M.M. Sylvestre M. J. Bacteriol. 2002; 184: PubMed Scopus Google In this work, we have used an that a of mutagenesis shuffling to a library of BPDO variants that were randomly mutated simultaneously on four residues of region of LB400 BphA. The of the was to the of that can in region III of of LB400 with of toward 2,2′-CB and the influence of new sequence on catalytic toward this and other chlorobiphenyls. of the variant of the library a rate of oxygenation of 2,2′-CB, regiospecificity toward this was The of and the metabolite of this variant toward a range of congeners were with obtained LB400 BPDO the The data suggest that the that residues and a influence on the regiospecificity toward 2,2′-CB and on the rate toward several congeners, the pattern of chlorine on the PCB substrate orientation toward the catalytic and M. J. 1992; Google was used in this in which bphA was mutated to an was D. H. Sylvestre M. J. Microbiol. 2001; Google it for a D. H. Sylvestre M. J. Microbiol. 2001; Google Scholar, Y. Barriault D. Sylvestre M. J. Bacteriol. PubMed Google the on BphA. was H. Y. Barriault D. Sylvestre M. J. Bacteriol. PubMed Google This for the ferredoxin and the ferredoxin reductase components of LB400 BPDO and for and 2,3-dihydroxybiphenyl from xenovorans which are the and of the biphenyl catabolic pathway. The reaction BPDO and biphenyl into was as a from LB400 and and then into the were to J. was by of and to BPDO and of which to were used with to of that the of that for region III (Fig. and 2 were used in with the 3 to the and that to the end portion of bphA and 2 were and to a to of the protein by other amino In was and 2 was The and to the encoding amino acid of the and extended from the of the that encoded for region III of to the end of the The other and were used in with to a that to the end portion of of and that extended from the of bphA to which to the end of region III of (Fig. and with and inside the that encoded region 4 was and to Phe336 of the 5 was and to Thr335 of the was and to of the was and to Thr335 and Phe336 and was and to Phe336 and The seven that inside region III were to generate a library of (Fig. The were with to the for shuffling PubMed Scopus Google was used for the The were as by that the was as for for for and for The was the and was as by The resulting library of was with and and the resulting were to from The library of was transformed into E. that the of to into were on on the of J. of the were to isopropyl the were for 3 and then of or of 2,2′-CB were into the of the is a that is normally rate similar or than biphenyl by BPDOs of natural LB400 BPDO H. M. PubMed Scopus Google It was used to the of the of the library an for which the that in region III did not to oxygenate this The were and to 2,2′-CB were a of to for that a than a LB400 BphA. to the PCB variant were for to a of a (6Barriault D. Plante M.M. Sylvestre M. J. Bacteriol. 2002; 184: PubMed Scopus Google The of of the 2,6-CB, 2,4,3′-CB, 2,4,4′-CB, 2,3,4′-CB, 2,6,2′,6′-CB, 2,5,2′,5′-CB, 2,3,2′,3′-CB, 2,4,2′,4′-CB, 2,4,3′,4′-CB, 3,4,3′,4′-CB, 3,4,3′,5′-CB, 2,3,4,2′,5′-CB, 2,4,5,3′,4′-CB, 2,4,5,2′,4′,5′-CB, 2,3,4,5,2′,5′-CB, and 5 of and The to not was a of The were obtained from of E. that were to the of the in the are from to the and the were from of of E. In this in were for 2 with and then and to an of in J. The was by of 2 in with of a of the were for with were with The were by gas chromatography-mass of or (6Barriault D. Plante M.M. Sylvestre M. J. Bacteriol. 2002; 184: PubMed Scopus Google Scholar, D. M. J. Sylvestre M. PubMed Scopus Google In the were by of E. D. M. J. Sylvestre M. PubMed Scopus Google a similar to the In the were from catalytic oxygenation BPDO from components that were obtained Y. Barriault D. Sylvestre M. J. Biol. Chem. 1996; 271: 8152-8156Abstract Full Text Full Text PDF PubMed Scopus (52) Google were from of substrate by Y. Barriault D. Sylvestre M. J. Bacteriol. PubMed Google for BPDO to randomly residues of region III we used the and in library of E. was were on and to the of a The was on the of by these In a first of we that of the were able to oxygenate the of were to 2,2′-CB into the of the variants that toward that a of toward of the library were to 2,2′-CB the seven a more than LB400 Based on the that variant a rate of reaction toward 2,2′-CB of from 2,2′-CB, we these for also that a of the a rather than the to The were to generate a metabolite that was to these variants were to have regiospecificity toward 2,2′-CB it established that catalytic oxygenation of this by LB400 BPDO as major metabolite J.D. Gibson D.T. J. Bacteriol. 1995; 177: PubMed Google Scholar, M. J. Bacteriol. 2001; PubMed Scopus Google that can cleaved by BphC. variants were also of the variants were for to a of PCB congeners. Based on J.D. Gibson D.T. J. Bacteriol. 1995; 177: PubMed Google Scholar, Haddock J.D. Appl. Environ. Microbiol. PubMed Scopus Google Scholar, M. Appl. Microbiol. 1995; PubMed Google Scholar, M. M. Appl. Environ. Microbiol. PubMed Google Scholar), with the of and 2,3,2′,3′-CB, of the are a rate by of LB400 BPDO or by E. this this the of these congeners by E. LB400 BPDO was than of of were to and were not The of congeners did not 2,2′-CB it was not from by was in other degrading potency of of are the of of PCB of a of to a of E. of the The is The are the of from three in The was of the in The for was to 2,6,2′,6′-CB, 3,4,3′,4′-CB, 2,4,5,3′,4′-CB, and were also in the PCB are not on the was and were was the and is also not in the and and of not The are the of of PCB of a of to a of E. of the The is The are the of from three in The was of the in The for was to 2,6,2′,6′-CB, 3,4,3′,4′-CB, 2,4,5,3′,4′-CB, and were also in the PCB are not on the was and were was the and is also not in the of not in a new of were to 2,2′-CB a rate than LB400 of of these E. 2,2′-CB from to with for E. LB400 of these that of were or for which the amino acid sequence of region III were or similar to the of variants obtained by Mondello F.J. B.D. Appl. Environ. Microbiol. PubMed Google variants and the sequence pattern variants and the sequence pattern and variants and the sequence pattern to the range of PCB substrates the data of Mondello F.J. B.D. Appl. Environ. Microbiol. PubMed Google the of Thr335 by a amino acid or did not the range of PCB congeners used as substrate Replacement of Thr335-Phe336 simultaneously by created variant which was able to oxygenate more efficiently congeners as and F.J. B.D. Appl. Environ. Microbiol. PubMed Google In the variant was able to oxygenate efficiently the congeners as that LB400 BPDO of the of the for to 2,2′-CB into a were was a in which was by was a Thr335-Phe336 was by It is noteworthy that of the seven and were that an amino acid sequence pattern Thr335 was by Ala and Phe336 was by E. BPDO 2,2′-CB a rate of which was similar to that of LB400 The rate of of 2,2′-CB by BPDO the rate of of 2,2′-CB by of variant that specific toward 2,2′-CB was in the range of 2,2′-CB with of for LB400 The specific of of BPDO was than that of LB400 biphenyl was the of biphenyl BPDO were with LB400 the rate of of biphenyl for this It was also the for several other PCB congeners that were oxygenated more efficiently by E. BPDO than LB400 BPDO the other BPDO oxygenated more efficiently than LB400 BPDO, the range of was than for variant and of 2,2′-CB by BPDO J.D. Gibson D.T. J. Bacteriol. 1995; 177: PubMed Google that were generated from oxygenation of 2,2′-CB by LB400 The major of the was as In a D. M. Sylvestre M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google we the metabolite as the variant was used to the the of the from 2,2′-CB from the obtained with LB400 E. BPDO of and of from 2,2′-CB In was a of a which was to D. M. Sylvestre M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google of variant BPDOs of to were from the metabolite in the of E. the BPDO and are on the the of metabolite obtained from and and and and The were from the metabolite in the of E. the BPDO and are on the the of metabolite obtained from in a new The of to was for of the and variants obtained in this The metabolite profile of variants was similar to that of LB400 BPDO However, it for of the variants that generated a of than LB400 other than and were from 2,2′-CB by of these of PCB by III and 4 the metabolite profile obtained from LB400 and BPDOs were used to the The were by in with the data in the The also shows the rate of oxygenation of these congeners by BPDO with that of LB400 generated by LB400 and BPDO from of to LB400 of BPDO to LB400 BPDO was from the of the of the of from 2 of with of on of generated by LB400 or metabolite of the on M. M. Appl. Environ. Microbiol. PubMed Google on M. M. Appl. Environ. Microbiol. PubMed Google on M. M. Appl. Environ. Microbiol. PubMed Google on M. M. Appl. Environ. Microbiol. PubMed Google on J.D. Gibson D.T. J. Bacteriol. 1995; 177: PubMed Google and Haddock J.D. Appl. Environ. Microbiol. PubMed Scopus Google an on carbons 3 and on D. M. Sylvestre M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google were obtained with E. LB400 or not metabolite with LB400 were obtained with E. LB400 or of the on carbons 2 and The of BPDO to LB400 BPDO was from the of the of the of from 2 of with of Based on of generated by LB400 or metabolite of the were obtained with E. LB400 or not metabolite with LB400 of the in a new LB400 BPDO J.D. Gibson D.T. J. Bacteriol. 1995; 177: PubMed Google Scholar, M. Appl. Microbiol. 1995; PubMed Google are of of 5 and or carbons 4 and In M. M. Appl. Environ. Microbiol. PubMed Google Scholar), on the of generated from by the four of the biphenyl catabolic was as the major and was as the This was of BPDO a metabolite from and were to the major metabolite from LB400 BPDO and this metabolite is to by M. Sylvestre M. D. Appl. Environ. Microbiol. 1992; PubMed Google the as LB400 BPDO in of was used to the the the of generated by BPDO 2 of was than of by of LB400 BPDO the the that in BPDO did not change the regiospecificity toward 2,4,3′-CB, 2,3,4′-CB, 2,4,4′-CB, and 2,4,2′,4′-CB, the rate of conversion of these congeners was was used to the reaction metabolite was from by both enzymes. Based on M. M. Appl. Environ. Microbiol. PubMed Google it was as were from not However, on the M. M. Appl. Environ. Microbiol. PubMed Google was as that a portion of the substrate was oxygenated on the ortho-meta carbons of the LB400 BPDO oxygenated on carbons 2 and 3 of the generated that not from However, the metabolite were the oxygenation was by LB400 or were also obtained from was not an metabolite profile was obtained for both enzymes. The metabolite profile and rate of of the congeners 2,5,2′,5′-CB, 2,3,2′,3′-CB and were the for both as as rate of Based on Haddock J.D. Appl. Environ. Microbiol. PubMed Scopus Google Scholar, M. M. Appl. Environ. Microbiol. PubMed Google Scholar), was oxygenated on carbons 3 and 4 to generate The from 2,3,2′,3′-CB by LB400 BPDO have D. M. Sylvestre M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google as and in a of that oxygenation on the vicinal meta-para carbons 4 and 5 is (Fig. Based on of the were in by BPDO to M. M. Appl. Environ. Microbiol. PubMed Google that was by LB400 BPDO, the rate of was not was used to metabolite was and it to a Based on the that and 2,3,2′,3′-CB J.D. Gibson D.T. J. Bacteriol. 1995; 177: PubMed Google Scholar, D. M. Sylvestre M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google are oxygenated principally on meta-para the metabolite is to this to The and the are not oxygenated by of LB400 BPDO or E. the metabolite as a was E. BPDO were used to the oxygenation of It noteworthy that this metabolite was the substrate was a than in the Appl. Environ. Microbiol. PubMed Google have the that the more PCB congeners are more efficiently is for this substrate of BPDO reaction by of to substrate for several J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google variant the to oxygenate this the of not it is noteworthy that the oxygenation not on the ortho-meta carbons 5 and the metabolite have E. BPDOs a metabolite from in a a conversion yield of of the The of were of a However, we were to the oxygenation on the or to congeners, were LB400 BPDO or E. this were used to the oxygenation of 2,6-CB, data (6Barriault D. Plante M.M. Sylvestre M. J. Bacteriol. 2002; 184: PubMed Scopus Google Scholar, M. Appl. Microbiol. 1995; PubMed Google three from the that were generated by variant (6Barriault D. Plante M.M. Sylvestre M. J. Bacteriol. 2002; 184: PubMed Scopus Google the of the not the that the metabolite of that variant BPDO oxygenated a portion of on vicinal ortho-meta carbons of the This was on the that did not an ortho-meta oxygenation on the of the The data suggest that structural that influence the regiospecificity toward 2,2′-CB are not the as that the regiospecificity toward the In this we have used the in to the to regiospecificity and substrate of amino acid residues of BPDO α subunit region was up to for variants rate or regiospecificity toward with LB400 BPDO, several of the variants of of several PCB congeners. In is a to structural to variants on the of the of the from 2,2′-CB were similar or to obtained F.J. B.D. Appl. Environ. Microbiol. PubMed Google by residues of LB400 by of The data suggest that the of amino acid of region III to the rate of oxygenation of 2,2′-CB by change of regiospecificity are rather The of sequence of region III of variants a change in regiospecificity toward 2,2′-CB was also of the three sequence an of the rate of toward a range of chlorobiphenyls. BPDOs to the oxygenation of is a The to to the oxygenation of an of which is by the of chlorine Thus congeners can a the that are on the can Regiospecificity toward is by chlorine between the chlorine and specific amino acid residues of the The of these amino acid residues and interactions with congeners is However, several amino acids of the C-terminal portion of have as for rate and regiospecificity toward congeners. the amino acids of region III of and especially of LB400 BPDO have to influence the substrate rate toward several congeners and the regiospecificity toward 2,2′-CB (6Barriault D. Plante M.M. Sylvestre M. J. Bacteriol. 2002; 184: PubMed Scopus Google Scholar, F.J. B.D. Appl. Environ. Microbiol. PubMed Google these amino acids the orientation that 2,2′-CB inside the catalytic and in it can change the of the biphenyl by this and a H. M. J. Bacteriol. 2002; 184: PubMed Scopus Google shows that and especially are in and Phe336 by Met or Ile as in or variants in a change in regiospecificity toward H. M. J. Bacteriol. 2002; 184: PubMed Scopus Google that a variant of BPDO in which to Phe336 of LB400 was by regiospecificity toward of the variants of that a oxygenation Thus the of LB400 BPDO the regiospecificity toward 2,2′-CB, and to a in this Based on of H. M. J. Bacteriol. 2002; 184: PubMed Scopus Google have a of that of BPDO, which to Phe336 of LB400 BPDO, is to the This as as is to an in The by which these residues with the substrate to influence and orientation of the substrate toward the catalytic is However, data that the amino acids that are these in are not that the structural to an extended range of PCB congeners Ala335-Met336 variants were in this the were major between variants and with to of toward chlorobiphenyls. the congeners The PCB degrading potency of variants the sequence pattern Mondello F.J. B.D. Appl. Environ. Microbiol. PubMed Google have the PCB degrading potency of a variant an sequence This variant the to oxygenate and other congeners. Based on these in to residues and able to the rate of oxygenation of a range of chlorobiphenyls. of the regiospecificity toward 2,2′-CB of variant with that of variant that this The variants regiospecificity toward 2,2′-CB oxygenation of carbons 3 and this it is not was the by 2,2′-dichlorobiphenyl BPDO ortho-meta of the phenyl the catalytic toward a range of PCB congeners was in and it is that the regiospecificity toward congeners other than 2,2′-CB was the for both and LB400 It is especially to that the that the of the biphenyl ring was not the of of 2,2′-CB by BPDO, the metabolite obtained from 2,4,2′,4′-CB was the as the obtained LB400 BPDO the and it was as a the other 2,3,2′,3′-CB was oxygenated principally on the meta-para carbons by LB400 The that in did not the regiospecificity of the toward this It is noteworthy that the congeners and 2,3,2′,3′-CB are both oxygenated principally on meta-para carbons by both LB400 and data suggest that the that residues and a influence on the regiospecificity toward 2,2′-CB and on the rate toward several congeners, the pattern of chlorine on the PCB substrate orientation toward the catalytic will to for the of the to the residues of region III with congeners to influence rate of oxygenation for of of However, by with J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), it is that interactions or between the chlorine substitutes on the biphenyl and specific amino acid residues of the protein influence the orientation of the biphenyl ring inside the catalytic pocket. The of the amino acid residues involved in these interactions are to to the range of PCB congeners that the can oxygenate

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