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 b iologically active steroid hormones. We previously reported the isolat ion of three distinct mouse cDNAs for 3betaHSD (3betaHSD I, II, and II I) and tissue-specific expression of their mRNAs. 3betaHSD I is expres sed only in gonads and adrenal glands, and 3betaHSD Il and III are exp ressed in both liver and kidneys. In the current study, we present dat a which demonstrate that transiently expressed 3betaHSD I and 3betaHSD III proteins can catalyze the conversion of the DELTA5-steroids, preg nenolone and dehydroepiandrosterone, to their respective DELTA4-3-keto steroids, progesterone and androstenedione. They also can dehydrogenat e the 3beta-hydroxy group of the 5alpha-reduced steroid 5alpha-androst anediol to yield dihydrotestosterone in the presence of the cofactor N AD+. The K(m) values of the expressed 3betaHSD I (for each of these su bstrates) were all below 0.2 muM. K(M) values of 3betaHSD III were gre ater for all substrates, with the greatest increase observed for pregn enolone, which was over 10-fold greater. Both forms of expressed prote in can catalyze the reduction of dihydrotestosterone to 5alpha-androst anediol in the presence of the cofactor NADH, but with considerably hi gher K(m) values (5.5 muM for form I and 6.8 muM for form III). The ob served maximum velocity of form I was much higher for all substrates e xamined. RNase protection and immunoblot analysis of expressed 3betaHS D I and III indicate that the difference in maximum velocity reflect d ifferences in the steady state levels of mRNA and amounts of protein. In addition, the expressed 3betaHSD III protein analyzed by Western bl ot has a lower mobility than the 3betaHSD I protein, both similar in m ol wt to the 3betaHSD proteins detected in mouse liver and adrenal gla nds, 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 hom ologous human 3betaHSD isoform could play an important role in cases o f genetic deficiency of the gonadal and adrenal isoform.