Clarke T R, Bain P A, Sha L, Payne A H
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0278.
Endocrinology. 1993 May;132(5):1971-6. doi: 10.1210/endo.132.5.8477648.
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.
3β-羟基类固醇脱氢酶/δ5-δ4-异构酶（3βHSD）催化δ5-3β-羟基类固醇向δ4-3-酮类固醇的转化，这是所有生物活性甾体激素生物合成中的关键步骤。我们之前报道了三种不同的小鼠3βHSD cDNA（3βHSD I、II和III）的分离及其mRNA的组织特异性表达。3βHSD I仅在性腺和肾上腺中表达，3βHSD II和III在肝脏和肾脏中均有表达。在本研究中，我们提供的数据表明，瞬时表达的3βHSD I和3βHSD III蛋白能够催化δ5-类固醇孕烯醇酮和脱氢表雄酮分别转化为其相应的δ4-3-酮类固醇孕酮和雄烯二酮。在辅因子NAD+存在的情况下，它们还能使5α-还原类固醇5α-雄烷二醇的3β-羟基脱氢生成双氢睾酮。表达的3βHSD I（针对每种底物）的Km值均低于0.2μM。3βHSD III对所有底物的Km值更高，孕烯醇酮的Km值增加最为显著，超过了10倍。在辅因子NADH存在的情况下，两种形式的表达蛋白都能催化双氢睾酮还原为5α-雄烷二醇，但Km值相当高（I型为5.5μM，III型为6.8μM）。对于所有检测的底物，观察到的I型最大反应速度要高得多。对表达的3βHSD I和III进行核糖核酸酶保护和免疫印迹分析表明，最大反应速度的差异反映了mRNA稳态水平和蛋白量的差异。此外，通过蛋白质印迹分析的表达的3βHSD III蛋白的迁移率低于3βHSD I蛋白，二者分子量分别与在小鼠肝脏和肾上腺中检测到的3βHSD蛋白相似。这些数据表明，在肝脏和肾脏中表达的3βHSD同工型具有将δ5-3β-羟基类固醇转化为δ4-3-酮类固醇的能力。数据表明，同源的人类3βHSD同工型在性腺和肾上腺同工型基因缺陷的情况下可能起重要作用。
