Purification and properties of cytochrome P-450 (P-450lun) catalyzing steroid 11 beta-hydroxylation in Curvularia lunata.
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.
Suzuki
K
K
Department of Chemistry, Faculty of Science, Kanazawa University, Japan.
Sanga
K
K
Chikaoka
Y
Y
Itagaki
E
E
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
D013252:Steroid 11-beta-Hydroxylase