title: Purification and properties of cytochrome P-450 (P-450lun) catalyzing steroid 11 beta-hydroxylation in Curvularia lunata.
abstract: Addition of 11-deoxycortisol to the culture medium of Curvularia lunata induced the increase of cytochrome P-450 content and steroid 11 beta-hydroxylase activity. The enzyme in cell-free extract produces cortisol from 11-deoxycortisol in the presence of NADPH and O2. The enzyme was partially stabilized by glycerol, 11-deoxycortisol, GSH and PMSF. The hydroxylation activity was strongly inhibited by carbon monooxide and sulfhydryl reagents. Cytochrome P-450 located on the microsomal fraction was solubilized with Triton X-100 and sodium cholate and purified to apparent homogeneity by column chromatography. The purified cytochrome P-450 (P-450lun) has a molecular mass of 60 kDa and exhibits the absorption maximum at 392 nm in the spectrum of oxidized form in the presence of 11-deoxycortisol. The reduced CO difference spectrum has a maximal peak at 448 nm. 11 beta-Hydroxylation of 11-deoxycortisol was reconstituted by cytochrome P-450lun, C. lunata NADPH-cytochrome P-450 reductase and DLPC in the presence of NADPH and O2 with a turnover number of 207 nmol/min per nmol of cytochrome P-450. The reductase and DLPC could be partially replaced with the enzyme purified from yeast or pig testis microsome and lipids purified from C. lunata, respectively. P-450lun catalyzes bifunctionally 11 beta- and 14 alpha-hydroxylations of 11-deoxycortisol. Deoxycorticosterone, progesterone, androstenedione and testosterone are hydroxylated in the similar manner.
authors.affiliation: Department of Chemistry, Faculty of Science, Kanazawa University, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Suzuki
authors.forename: K
authors.initials: K
authors.lastname: Sanga
authors.forename: Y
authors.initials: Y
authors.lastname: Chikaoka
authors.forename: E
authors.initials: E
authors.lastname: Itagaki
mesh_terms: D000818:Animals; D002384:Catalysis; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D003904:Mitosporic Fungi / Q000201:enzymology*; D013057:Spectrum Analysis; D013252:Steroid 11-beta-Hydroxylase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D013252:Steroid 11-beta-Hydroxylase