Involvement of the Terminal Oxygenase β Subunit in the Biphenyl Dioxygenase Reactivity Pattern toward Chlorobiphenyls

Abstract

ABSTRACT 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 (α and β) iron sulfur protein (ISP BPH ), a ferredoxin (FER BPH ), and a ferredoxin reductase (RED BPH ). 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 ISP BPH α or β 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 α subunit of ISP BPH of strain LB400 and the β subunit of ISP BPH of strain B-356 (the α LB400 β B-356 chimera) and the α B-356 β LB400 chimera, were functional. Results with purified enzyme preparations showed for the first time that the ISP BPH β subunit influences BPH dox’s reactivity pattern toward chlorobiphenyls. Thus, if the α subunit were the sole determinant of the enzyme reactivity pattern, the α B-356 β LB400 chimera should have behaved like B-356 ISP BPH ; instead, its reactivity pattern toward the substrates tested was similar to that of LB400 ISP BPH . On the other hand, the α LB400 β B-356 chimera showed features of both B-356 and LB400 ISP BPH 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.