title: Purification and characterization of NADPH-cytochrome P450 reductase from filamentous fungus Rhizopus nigricans.
abstract: We report here the isolation and partial characterization of a flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase. The enzyme is a part of steroid 11 alpha-hydroxylating system and is associated with the microsomal fraction of the fungus Rhizopus nigricans. Fungal reductase was solubilized from microsomal membranes with Triton X-100 and purified to apparent homogeneity by affinity and high-performance ion-exchange chromatography. A 350-fold purification of the enzyme with specific activity of 37 mumol cytochrome c reduced/min/mg protein was achieved. A single protein band was obtained on SDS-PAGE analysis with an apparent molecular weight of 79 kDa. Purified reductase contained approximately equimolar quantities of flavin adenine dinucleotide and flavin mononucleotide per mole of the enzyme. Upon induction of the steroid hydroxylating system with progesterone the activity of microsomal NADPH-cytochrome c (P450) reductase increased 10-fold. This is in good correlation with the increase in content of fungal cytochrome P450. Purified fungal flavoprotein was active in a reconstituted system with cytochrome P450 C21 from adrenal gland but could not replace adrenodoxin reductase in the mitochondrial steroid 11 beta-hydroxylating system. We were able to confirm the role of the enzyme by reconstituting steroid 11 alpha-hydroxylating activity from the separated components NADPH-cytochrome P450 reductase and cytochrome P450, partly purified from fungal microsomes.
authors.affiliation: Institute of Biochemistry, Medical Faculty, University of Ljubljana, Slovenia.
authors.forename: T
authors.initials: T
authors.lastname: Makovec
authors.forename: K
authors.initials: K
authors.lastname: Breskvar
mesh_terms: D003067:Coenzymes / Q000502:physiology; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction; D004795:Enzyme Stability; D006900:Hydroxylation / Q000187:drug effects; D007424:Intracellular Fluid / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D009251:NADPH-Ferrihemoprotein Reductase; D012233:Rhizopus / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D003067:Coenzymes; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase