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 FERBPH, and a ferredoxin reductase REDBPH. REDBPH and FERBPH 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 alpha3beta3 heteromer, with each alphabeta unit containing a [2Fe-2S] Rieske center (epsilon455 = 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.
Hurtubise
Y
Y
INRS-Santé, Institut National de la Recherche Scientifique, Pointe-Claire, Québec, H9R 1G6 Canada.
Barriault
D
D
Sylvestre
M
M
D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D006088:Gram-Negative Aerobic Bacteria / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry; D010105:Oxygenases / Q000737:chemistry*; D011994:Recombinant Proteins; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
D017931:DNA Primers; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D006639:Histidine; D010105:Oxygenases