Catalytic and immunochemical properties of NADPH-cytochrome P450 reductase from fungus Rhizopus nigricans

• Published in: The Journal of steroid biochemistry and molecular biology Vol. 82; no. 1; pp. 89 - 96
• Main Authors: Makovec, Tomaž, Breskvar, Katja
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.09.2002; Elsevier Science
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Blotting, Western; Catalysis; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System - metabolism; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Filamentous fungus; Fundamental and applied biological sciences. Psychology; Isoenzymes; Mice; Microsomes - enzymology; NADPH-cytochrome P450 reductase; NADPH-Ferrihemoprotein Reductase - antagonists & inhibitors; NADPH-Ferrihemoprotein Reductase - chemistry; NADPH-Ferrihemoprotein Reductase - immunology; NADPH-Ferrihemoprotein Reductase - metabolism; Oxidoreductases; Peptide Fragments - chemistry; Progesterone; Rhizopus - enzymology; Steroid Hydroxylases - antagonists & inhibitors; Steroid Hydroxylases - metabolism; Subcellular Fractions - enzymology; Trypsin - metabolism; NADPH-cytochrome P450 reductase; Filamentous fungus; Progesterone; Molecular form; Purification; Enzyme; Rhizopus stolonifer; Cytochrome P450; Phycomycetes; NADPH-ferrihemoprotein reductase; In vitro; Ovarian hormone; Fungi; Characterization; Oxidoreductases; Sex steroid hormone; Catalyst activity; Thallophyta
• ISSN: 0960-0760; 1879-1220
• DOI: 10.1016/S0960-0760(02)00145-0

Flavoprotein NADPH-cytochrome P450 reductase (CPR, EC 1.6.2.4) from filamentous fungus Rhizopus nigricans is a membrane bound enzyme which is involved in the reduction of cytochrome P450 during the hydroxylation of progesterone at 11α position. After purification of the enzyme from induced mycelia three forms of fungal CPR were detected on SDS-PAGE: a predominant form with an apparent molecular mass of 78 kDa and two truncated forms. N-terminal sequences of all three forms were determined as well as some internal sequences of 78 kDa form. Dose-dependent immunoinhibition of NADPH-cytochrome c reductase and progesterone 11α-hydroxylase activities was observed with mouse anti-CPR antisera. No cross-reactions were obtained on Western blots between mouse anti-CPR antisera and protein preparations from noninduced mycelia and microsomal fraction from fungus Pleurotus osteatus, plant Ginkgo biloba or chicken liver. The kinetic mechanism of CPR was proposed on the basis of model reaction with cytochrome c 3+. Results obtained at high ionic strength suggest a nonclassical two-site ping pong mechanism and at low ionic strength a sequential mechanism of bisubstrate reaction.