Characterization of active recombinant His-tagged oxygenase component of Comamonas testosteroni B-356 biphenyl dioxygenase

Biphenyl (BPH) dioxygenase oxidizes BPH to 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356, The enzyme comprises a two-subunit iron-sulfur protein (ISPBPH), a ferredoxin FER(BPH), and a ferredoxin reductase RED(BPH). RED(BPH) and FER(BPH) transfer electrons from NADH to an Fe-S active center of ISPBPH which activates molecular oxygen for insertion into the substrate, In this work B-356 ISPBPH complex and its alpha and beta subunits were purified from recombinant Escherichia coli strains using the His-bind QIAGEN system. His-tagged B-356 ISPBPH construction carrying a single His tail on the N-terminal portion of the alpha subunit was active. Its major features were compared to the untagged enzyme. In both cases, the native form is an alpha(3) beta(3) heteromer, with each alpha beta unit containing a [2Fe-2S] Rieske center (epsilon(455) = 8,300 M(-1) cm(-1)) and a mononuclear Fe2+. Although purified His-tagged alpha subunit showed the characteristic absorption spectra of Rieske-type protein, reassociation of this enzyme component and His-tagged beta subunit to reconstitute active ISPBPH was weak. However, when His-tagged alpha and beta subunits were reassembled in vitro in crude cell extracts from E. coli recombinants, active ISPBPH could be purified on Ni-nitrilotriacetic acid resin.