20beta-hydroxy-C21-steroid 20beta-oxidase activity of cytochrome P450c21 purified from bovine adrenocortical microsomes.
We showed previously that cytochrome P450c21 (CYP21A1) from bovine adrenocortical microsomes has a putative 20beta-oxidase activity for 20beta-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 20beta-oxidase activity of cytochrome P450c21. Combination of 17alpha, 20beta-dihydroxyprogesterone with purified cytochrome P450c21 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 microM. EPR spectrum at low temperature (15 K) exhibited an appearance of a new low-spin signal at gz=2.42, gy=2.21, and gx=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 17alpha-hydroxyprogesterone and 20beta-hydroxyprogesterone. The enzymatic activity for 17alpha, 20beta-dihydroxyprogesterone was confirmed in a reconstituted system consisting of cytochrome P450c21 and NADPH-cytochrome P450 reductase. 17alpha,20beta-Dihydroxyprogesterone was converted to 17alpha-hydroxyprogesterone via the 20beta-oxidase reaction. The high turnover numbers of the 20beta-oxidase activity for 20beta-hydroxy-c21-steroids suggests that this activity is likely to have some physiological roles. Cytochrome P450c21 and 20beta-hydroxysteroid dehydrogenase may regulate the androgen biosynthesis catalyzed by cytochrome P450c17alpha with controlling the concentration of 20beta-hydroxy-C21-steroids.
Tsubaki
M
M
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Kamigoori-cho, Akou-gun, Hyogo 678-12, Japan. tsubaki@sci.himeji-tech.ac.jp
Matsumoto
N
N
Tomita
S
S
Ichikawa
Y
Y
Hori
H
H
D000311:Adrenal Glands / Q000201:enzymology*; D000728:Androgens / Q000096:biosynthesis; D000818:Animals; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006908:Hydroxyprogesterones / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism*; D015394:Molecular Structure; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010088:Oxidoreductases / Q000378:metabolism; D013255:Steroid 21-Hydroxylase; D013256:Steroids / Q000032:analysis
D000728:Androgens; D006908:Hydroxyprogesterones; D013256:Steroids; C007433:17,20-dihydroxy-4-pregnen-3-one; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D013255:Steroid 21-Hydroxylase; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase