The 3-beta-hydroxysteroid dehydrogenase/ DELTA-5-DELTA-4 isomerase (3-beta-HSD) enzyme catalyzes the oxidation and isomerization of DELTA-5-3-beta-hydroxysteroid precursors into DELTA-4-ketosteroids, thus leading to the formation of all classes of steroid hormones. In addition, 3-beta-HSD catalyzes the interconversion of 3-beta-hydroxy- and 3-keto-5-alpha-androstane steroids. Clinical observations in patients with 3-beta-HSD deficiency as well as our recent data obtained by Southern blot analysis using a human placental 3-beta-HSD cDNA (type I) as probe suggested the existence of multiple related 3-beta-HSD isoenzymes. We now report the isolation and characterization of a second type of cDNA clone (arbitrarily designated type ll) encoding 3-beta-HSD after screening of a human adrenal lambda-gt22A library. The nucleotide sequence of 1676 basepairs of human 3-beta-HSD type II cDNA predicts a protein of 371 amino acids with a calculated molecular mass of 41,921 daltons, which displays 93.5 and 96.2% homology with human placental type l and rhesus macaque ovary 3-beta-HSD deduced proteins, respectively. To characterize and compare the kinetic properties of the two isoenzymes, plasmids derived from pCMV and containing type I or type II 3-beta-HSD full-length cDNA inserts were transiently expressed in HeLa human cervical carcinoma cells. In vitro incubation with NAD+ and H-3-labeled pregnenolone or dehydroepiandrosterone shows that the type I protein possesses a 3-beta-HSD/DELTA-5-DELTA-4 isomerase activity higher than type II, with respective K(m) values of 0.24 vs. 1.2-mu-M for pregnenolone and 0.18 vs. 1.6-mu-M for dihydroepiandrosterone, while the specific activity of both types is equivalent. Moreover, incubation in the presence of NADH of homogenates from cells transfected with type I or type II 3-beta-HSD indicates that dihydrotestosterone is converted into 5-alpha-androstane-3-beta, 17-beta-diol, with K(m) values of 0.26 and 2.7-mu-M, respectively. Ribonuclease protection assay using type I- and type II-specific cRNA probes revealed that type II transcripts are the almost exclusive 3-beta-HSD mRNA species in the human adrenal gland, ovary, and testis, while type I transcripts correspond to the almost exclusive 3-beta-HSD mRNA species in the placenta and skin and represent the predominantly expressed species in mammary gland tissue. The present data show for the first time that adrenals and gonads express a type of 3-beta-HSD isoenzyme that is distinct from the type expressed in the placenta. Detection of 3-beta-HSD gene expression in nonclassical steroidogenic tissues such as skin and mammary gland suggests that 3-beta-HSD is likely to play an important role in the intracrine formation of sex steroids in peripheral target tissues. Characterization of type II 3-beta-HSD, in addition to permitting studies of the regulation of its tissue-specific expression, offers the opportunity of elucidating the molecular basis of classical and nonclassical 3-beta-HSD deficiencies, the second most common cause of congenital adrenal hyperplasia and the predicted most common genetic disorder in women with signs of androgen excess, respectively.
