ENZYME CHARACTERISTICS OF 2 DISTINCT FORMS OF MOUSE 3-BETA-HYDROXYSTEROID DEHYDROGENASE/DELTA-5-DELTA-4-ISOMERASE COMPLEMENTARY DEOXYRIBONUCLEIC ACIDS EXPRESSED IN COS-1 CELLS

The enzyme 3beta-hydroxysteroid dehydrogenase/DELTA5-DELTA4-Isomerase (3betaHSD) catalyzes the conversion of DELTA5-3beta-hydroxysteroids to DELTA4-3-ketosteroids, an essential step in the biosynthesis of all biologically active steroid hormones. We previously reported the isolation of three distinct mouse cDNAs for 3betaHSD (3betaHSD I, II, and III) and tissue-specific expression of their mRNAs. 3betaHSD I is expressed only in gonads and adrenal glands, and 3betaHSD Il and III are expressed in both liver and kidneys. In the current study, we present data which demonstrate that transiently expressed 3betaHSD I and 3betaHSD III proteins can catalyze the conversion of the DELTA5-steroids, pregnenolone and dehydroepiandrosterone, to their respective DELTA4-3-ketosteroids, progesterone and androstenedione. They also can dehydrogenate the 3beta-hydroxy group of the 5alpha-reduced steroid 5alpha-androstanediol to yield dihydrotestosterone in the presence of the cofactor NAD+. The K(m) values of the expressed 3betaHSD I (for each of these substrates) were all below 0.2 muM. K(M) values of 3betaHSD III were greater for all substrates, with the greatest increase observed for pregnenolone, which was over 10-fold greater. Both forms of expressed protein can catalyze the reduction of dihydrotestosterone to 5alpha-androstanediol in the presence of the cofactor NADH, but with considerably higher K(m) values (5.5 muM for form I and 6.8 muM for form III). The observed maximum velocity of form I was much higher for all substrates examined. RNase protection and immunoblot analysis of expressed 3betaHSD I and III indicate that the difference in maximum velocity reflect differences in the steady state levels of mRNA and amounts of protein. In addition, the expressed 3betaHSD III protein analyzed by Western blot has a lower mobility than the 3betaHSD I protein, both similar in mol wt to the 3betaHSD proteins detected in mouse liver and adrenal glands, respectively. These data demonstrate that an isoform of 3betaHSD expressed in liver and kidney has the capacity to convert DELTA5-3beta-hydroxysteroids to DELTA4-3-ketosteroids. The data suggest that a homologous human 3betaHSD isoform could play an important role in cases of genetic deficiency of the gonadal and adrenal isoform.