20-BETA-HYDROXY-C21-STEROID 20-BETA-OXIDASE ACTIVITY OF CYTOCHROME P450(C21) PURIFIED FROM BOVINE ADRENOCORTICAL MICROSOMES

We showed previously that cytochrome P450(c21) (CYP21A1) from bovine a drenocortical microsomes has a putative 20 beta-oxidase activity for 2 0 beta-hydroxyprogesterone leading to a conversion to progesterone [M. Tsubaki, K. Morimoto, S, Tomita, S. Miura, Y, Ichikawa, A. Miyatake, F. Masuya, H. Hori, Biochim. Biophys. Acta 1259 (1995) 89-98]. We have extended the investigation on the 20 beta-oxidase activity of cytochr ome P450(c21). Combination of 17 alpha,20 beta-dihydroxyprogesterone w ith purified cytochrome P450(c21) in oxidized form caused an induction of a typical type I difference spectrum (a peak at 390 nm and a troug h at 420 nm) with a spectral dissociation constant of 2.3 mu M. EPR sp ectrum at low temperature (15 K) exhibited an appearance of a new low- spin signal at g(z) = 2.42, g(y) = 2.21, and g(x) = 1.966 and an incre ase in intensity of the high-spin signal (g = 8.06, 3.54) upon formati on of the enzyme-steroid complex, as previously found for 17 alpha-hyd roxyprogesterone and 20 beta-hydroxyprogesterone. The enzymatic activi ty for 17 alpha,20 beta-dihydroxyprogesterone was confirmed in a recon stituted system consisting of cytochrome P450(c21) and NADPH-cytochrom e P450 reductase, 17 alpha,20 beta-Dihydroxyprogesterone was converted to 17 alpha-hydroxyprogesterone via the 20 beta-oxidase reaction. The high turnover numbers of the 20 beta-oxidase activity for 20 beta-hyd roxy-c21-steroids suggests that this activity is likely to have some p hysiological roles, Cytochrome P450(c21) and 20 beta-hydroxysteroid de hydrogenase may regulate the androgen biosynthesis catalyzed by cytoch rome P450(c17 alpha) with controlling the concentration of 20 beta-hyd roxy-C21-steroids. (C) 1998 Elsevier catalyzed by cytochrome Science B .V.