Purification and properties of cytochrome P-450 ( P-450 lun) catalyzing steroid 11β-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 11β-hydroxylase activity. The enzyme in cell-free extract produces cortisol from 11-deoxycortisol in the presence of NADPH and O 2. The enzyme was partially stabilized by glycerol, 11-deoxycortisol, GSH and PMSF. The hydroxylation activity was strongly inhibited by carbon monooxid 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-450 lun) has a molecular mass of 60 kDa and exhibits the absorption maxium 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. 11β-Hydroxylation of 11-deoxycortisol was reconstituted by cytochrome P-450 lun, C. lunata NADPH-cytochrome P-450 reductase and DLPC in the presence of NADPH and O 2 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-450 lun catalyzes bifunctionally 11β- and 14α-hydroxylations of 11-deoxycortisol. Deoxycorticosterone, progesterone, androstenedione and testosterone are hydroxylated in the similar manner.