INVOLVEMENT OF THE TERMINAL OXYGENASE BETA-SUBUNIT IN THE BIPHENYL DIOXYGENASE REACTIVITY PATTERN TOWARD CHLOROBIPHENYLS

Biphenyl dioxygenase (BPH dox) oxidizes biphenyl on adjacent carbons t o 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 preferenti ally catalyzes the oxidation of the double-meta-substituted congener 3 ,3'-dichlorobiphenyl over the double-para-substituted congener 4,4'-di chlorobiphenyl or the double-ortho-substituted congener 2,2'-dichlorob iphenyl. LB400 BPH dos shows a preference for 2,2'-dichlorobiphenyl, a nd in addition, unlike B-356 BPH dox, it can catalyze the oxidation of selected chlorobiphenyls such as 2,2',5,5'-tetrachlorobiphenyl on adj acent meta-para carbons. In this work, we examine the reactivity patte rn of BPH dox toward various chlorobiphenyls and its capacity to catal yze the meta-pam dioxygenation of chimeric enzymes obtained by exchang ing the ISPBPH alpha or beta subunit of strain B-356 for the correspon ding subunit of strain LB400. These hybrid enzymes were purified by an affinity chromatography system as His-tagged proteins. Both types, th e chimera with the alpha subunit of ISPBPH of strain LB400 and the bet a subunit of ISPBPH of strain B-356 (the alpha(LB400)beta(B-356) chime ra) and the alpha(B-356)beta(LB400) chimera, were functional. Results with purified enzyme preparations showed for the first time that the I SPBPH beta subunit influences BPH dox's reactivity pattern toward chlo robiphenyls. Thus, if the alpha subunit were the sole determinant of t he enzyme reactivity pattern, the alpha(B-356)beta(LB400) chimera shou ld have behaved like B-356 ISPBPH; instead, its reactivity pattern tow ard the substrates tested was similar to that of LB400 ISPBPH. On the other hand, the alpha(LB400)beta(B-356) chimera showed features of bot h 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.