PURIFICATION AND CHARACTERIZATION OF THE COMAMONAS-TESTOSTERONI B-356BIPHENYL DIOXYGENASE COMPONENTS

In this report, we describe some of the characteristics of the Comamon as testosteroni B-356 biphenyl (BPH)chlorobiphenyl dioxygenase system, which includes the terminal oxygenase, an iron-sulfur protein (ISPBPH ) made up of an ol subunit (51 kDa) and a beta subunit (22 kDa) encode d by bphA and bphE, respectively; a ferredoxin (FER(BPH); 12 kDa) enco ded by bphF; and a ferredoxin reductase (RED(BPH); 43 kDa) encoded by bphG. ISPBPH subunits were purified from B-356 cells grown on BPH, Sin ce highly purified FER(BPH) and RED(BPH) were difficult to obtain from strain B-356, these two components were purified from recombinant Esc herichia coli strains by using the His tag purification system, These His-tagged fusion proteins were shown to support BPH 2,3-dioxygenase a ctivity in vitro when added to preparations of ISPBPH in the presence of NADH. FER(BPH) and RED(BPH) are thought to pass electrons from NADH to ISPBPH, which then activates molecular oxygen for insertion into t he aromatic substrate. The reductase was found to contain approximatel y 1 mol of flavin adenine dinucleotide per mol of protein and was spec ific for NADH as an electron donor, The ferredoxin was found to contai n a Rieske-type [2Fe-2S] center (epsilon(460) 7,455 M(-1) cm(-1)) whic h was readily lost from the protein during purification and storage. I n the presence of RED(BPH) and FER(BPH), ISPBPH, was able to convert B PH into both 2,3-dihydro-2,3-dihydroxybiphenyl and 3,4-dihydro-3,4-dih ydroxybiphenyl. The significance of this observation is discussed.