CHARACTERIZATION OF ACTIVE RECOMBINANT HIS-TAGGED OXYGENASE COMPONENTOF COMAMONAS-TESTOSTERONI B-356 BIPHENYL DIOXYGENASE

Biphenyl (BPH) dioxygenase oxidizes BPH to 2,3-dihydro-2,3-dihydroxybi phenyl in Comamonas testosteroni B-356, The enzyme comprises a two-sub unit iron-sulfur protein (ISPBPH), a ferredoxin FER(BPH), and a ferred oxin reductase RED(BPH). RED(BPH) and FER(BPH) transfer electrons from NADH to an Fe-S active center of ISPBPH which activates molecular oxy gen for insertion into the substrate, In this work B-356 ISPBPH comple x and its alpha and beta subunits were purified from recombinant Esche richia coli strains using the His-bind QIAGEN system. His-tagged B-356 ISPBPH construction carrying a single His tail on the N-terminal port ion 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] Rie ske 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 enzym e component and His-tagged beta subunit to reconstitute active ISPBPH was weak. However, when His-tagged alpha and beta subunits were reasse mbled in vitro in crude cell extracts from E. coli recombinants, activ e ISPBPH could be purified on Ni-nitrilotriacetic acid resin.