Enzyme characteristics of two distinct forms of mouse 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase complementary deoxyribonucleic acids expressed in COS-1 cells.
The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-Isomerase (3 beta HSD) catalyzes the conversion of delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids, an essential step in the biosynthesis of all biologically active steroid hormones. We previously reported the isolation of three distinct mouse cDNAs for 3 beta HSD (3 beta HSD I, II, and III) and tissue-specific expression of their mRNAs. 3 beta HSD I is expressed only in gonads and adrenal glands, and 3 beta HSD II and III are expressed in both liver and kidneys. In the current study, we present data which demonstrate that transiently expressed 3 beta HSD I and 3 beta HSD III proteins can catalyze the conversion of the delta 5-steroids, pregnenolone and dehydroepiandrosterone, to their respective delta 4-3-ketosteroids, progesterone and androstenedione. They also can dehydrogenate the 3 beta-hydroxy group of the 5 alpha-reduced steroid 5 alpha-androstanediol to yield dihydrotestosterone in the presence of the cofactor NAD+. The Km values of the expressed 3 beta HSD I (for each of these substrates) were all below 0.2 microM. Km values of 3 beta HSD 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 5 alpha-androstanediol in the presence of the cofactor NADH, but with considerably higher Km values (5.5 microM for form I and 6.8 microM for form III). The observed maximum velocity of form I was much higher for all substrates examined. RNase protection and immunoblot analysis of expressed 3 beta HSD 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 3 beta HSD III protein analyzed by Western blot has a lower mobility than the 3 beta HSD I protein, both similar in mol wt to the 3 beta HSD proteins detected in mouse liver and adrenal glands, respectively. These data demonstrate that an isoform of 3 beta HSD expressed in liver and kidney has the capacity to convert delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids. The data suggest that a homologous human 3 beta HSD isoform could play an important role in cases of genetic deficiency of the gonadal and adrenal isoform.
Clarke
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Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0278.
Bain
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Sha
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Payne
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D015113:Androstane-3,17-diol / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D003687:Dehydroepiandrosterone / Q000378:metabolism; D015870:Gene Expression; D015151:Immunoblotting; D007700:Kinetics; D051379:Mice; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D009243:NAD / Q000494:pharmacology; D011284:Pregnenolone / Q000378:metabolism; D011375:Progesterone Reductase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012260:Ribonucleases; D013251:Steroid Isomerases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
C021425:3 beta-hydroxysteroid oxidoreductase-delta(5) 3-ketosteroid isomerase; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D009243:NAD; D015113:Androstane-3,17-diol; D003687:Dehydroepiandrosterone; D011284:Pregnenolone; D004247:DNA; D011375:Progesterone Reductase; D012260:Ribonucleases; D013251:Steroid Isomerases