Evolution of the biphenyl dioxygenase BphA from Burkholderia xenovorans LB400 by random mutagenesis of multiple sites in region III.

Abstract 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.