20β-hydroxy-C21-steroid 20β-oxidase activity of cytochrome P450 c21 purified from bovine adrenocortical microsomes

Abstract

We showed previously that cytochrome P450 c21 (CYP21A1) from bovine adrenocortical microsomes has a putative 20β-oxidase activity for 20β-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 20β-oxidase activity of cytochrome P450 c21. Combination of 17α,20β-dihydroxyprogesterone with purified cytochrome P450 c21 in oxidized form caused an induction of a typical type I difference spectrum (a peak at 390 nm and a trough at 420 nm) with a spectral dissociation constant of 2.3 μM. EPR spectrum 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 increase in intensity of the high-spin signal ( g=8.06, 3.54) upon formation of the enzyme-steroid complex, as previously found for 17α-hydroxyprogesterone and 20β-hydroxyprogesterone. The enzymatic activity for 17α,20β-dihydroxyprogesterone was confirmed in a reconstituted system consisting of cytochrome P450 c21 and NADPH-cytochrome P450 reductase. 17α,20β-Dihydroxyprogesterone was converted to 17α-hydroxyprogesterone via the 20β-oxidase reaction. The high turnover numbers of the 20β-oxidase activity for 20β-hydroxy-c21-steroids suggests that this activity is likely to have some physiological roles. Cytochrome P450 c21 and 20β-hydroxysteroid dehydrogenase may regulate the androgen biosynthesis catalyzed by cytochrome P450 c17α with controlling the concentration of 20β-hydroxy-C21-steroids.