Molecular cloning of a novel type of rat cytoplasmic 17β-hydroxysteroid dehydrogenase distinct from the type 5 isozyme
Molecular cloning of a novel type of rat cytoplasmic 17β-hydroxysteroid dehydrogenase distinct from the type 5 isozyme
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DOI：
10.1093/jb/mvj109
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发表时间：
2006-06-01
影响因子：
2.7
通讯作者：
Hara, Akira
中科院分区：
文献类型：
--
作者：
Ishikura, Shuhei;Matsumoto, Kengo;Hara, Akira
大鼠肝脏含有两种细胞溶质酶（TBER 1和TBER 2），可将6-叔丁基-2，3-环氧-5-环己烯-1，4-二酮还原为其4 R-和4S-羟基代谢物。在这项研究中，我们克隆了TBER 1的cDNA，并使用同源重组酶检查内源性底物。该cDNA编码的蛋白质由323个氨基酸组成，属于醛酮还原酶家族。重组TBER 1在pH 7.4下使用NAD（+）作为优选的辅酶有效地氧化17 β-羟基类固醇和异生物质脂环醇，并且对20 α-和3 α-羟基类固醇以及9-羟基异生物素表现出低活性。己烯雌酚、己雌酚和玉米赤霉烯酮可有效抑制该酶。辅酶特异性，广泛的底物特异性和抑制剂的敏感性的酶不同的大鼠NADPH依赖性17 β-羟基类固醇脱氢酶5型，这是从肝脏中克隆，其特征在于使用重组酶。TBER 1的mRNA在大鼠肝脏、胃肠道和卵巢中高度表达，而17 β-羟基类固醇脱氢酶5型mRNA在肝脏和肾脏中特异性表达。因此，TBER 1代表了一种新型的17 β-羟基类固醇脱氢酶，具有独特的催化特性和组织分布。此外，TBER 2被确定为3 α-羟基类固醇脱氢酶的酶活性在大鼠肝细胞质中的色谱分析和重组3 α-羟基类固醇脱氢酶的表征。
Rat liver contains two cytosolic enzymes (TBER1 and TBER2) that reduce 6-tert-butyl-2,3-epoxy-5-cyclohexene-1,4-dione into its 4R- and 4S-hydroxy metabolites. In this study, we cloned the cDNA for TBER1 and examined endogenous substrates using the homogenous recombinant enzyme. The cDNA encoded a protein composed of 323 amino acids belonging to the aldo-keto reductase family. The recombinant TBER1 efficiently oxidized 17 beta-hydroxysteroids and xenobiotic alicyclic alcohols using NAD(+) as the preferred coenzyme at pH 7.4, and showed low activity towards 20 alpha- and 3 alpha-hydroxysteroids, and 9-hydroxyprostaglandins. The enzyme was potently inhibited by diethylstilbestrol, hexestrol and zearalenone. The coenzyme specificity, broad substrate specificity and inhibitor sensitivity of the enzyme differed from those of rat NADPH-dependent 17 beta-hydroxysteroid dehydrogenase type 5, which was cloned from the liver and characterized using the recombinant enzyme. The mRNA for TBER1 was highly expressed in rat liver, gastrointestinal tract and ovary, in contrast to specific expression of 17 beta-hydroxysteroid dehydrogenase type 5 mRNA in the liver and kidney. Thus, TBER1 represents a novel type of 17 beta-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution. In addition, TBER2 was identified as 3 alpha-hydroxysteroid dehydrogenase on chromatographic analysis of the enzyme activities in rat liver cytosol and characterization of the recombinant 3 alpha-hydroxysteroid dehydrogenase.