|
pubmed:abstractText |
Biphenyl dioxygenase (BPH dox) oxidizes biphenyl on adjacent carbons to generate 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356 and in Pseudomonas sp. strain LB400. The enzyme comprises a two-subunit (alpha and beta) iron sulfur protein (ISPBPH), a ferredoxin (FERBPH), and a ferredoxin reductase (REDBPH). B-356 BPH dox preferentially catalyzes the oxidation of the double-meta-substituted congener 3,3'-dichlorobiphenyl over the double-para-substituted congener 4,4'-dichlorobiphenyl or the double-ortho-substituted congener 2,2'-dichlorobiphenyl. LB400 BPH dox shows a preference for 2,2'-dichlorobiphenyl, and in addition, unlike B-356 BPH dox, it can catalyze the oxidation of selected chlorobiphenyls such as 2,2',5,5'-tetrachlorobiphenyl on adjacent meta-para carbons. In this work, we examine the reactivity pattern of BPH dox toward various chlorobiphenyls and its capacity to catalyze the meta-para dioxygenation of chimeric enzymes obtained by exchanging the ISPBPH alpha or beta subunit of strain B-356 for the corresponding subunit of strain LB400. These hybrid enzymes were purified by an affinity chromatography system as His-tagged proteins. Both types, the chimera with the alpha subunit of ISPBPH of strain LB400 and the beta subunit of ISPBPH of strain B-356 (the alphaLB400 betaB-356 chimera) and the alphaB-356betaLB400 chimera, were functional. Results with purified enzyme preparations showed for the first time that the ISPBPH beta subunit influences BPH dox's reactivity pattern toward chlorobiphenyls. Thus, if the alpha subunit were the sole determinant of the enzyme reactivity pattern, the alphaB-356betaLB400 chimera should have behaved like B-356 ISPBPH; instead, its reactivity pattern toward the substrates tested was similar to that of LB400 ISPBPH. On the other hand, the alphaLB400 betaB-356 chimera showed features of both B-356 and LB400 ISPBPH where the enzyme was able to metabolize 2,2'- and 3, 3'-dichlorobiphenyl and where it was able to catalyze the meta-para oxygenation of 2,2',5,5'-tetrachlorobiphenyl.
|
