Molecular characterization of a first human 3(α→β)-hydroxysteroid epimerase
Molecular characterization of a first human 3(α→β)-hydroxysteroid epimerase
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DOI：
10.1074/jbc.m000562200
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发表时间：
2000-09-22
影响因子：
4.8
通讯作者：
Van, LT
中科院分区：
文献类型：
--
作者：
Huang, XF;Van, LT
在本报告中，我们描述了编码酶的 cDNA 的分离和表征，该酶表现出 3(α-->β)-羟基类固醇差向异构酶 (3(α-->β)-HSE) 的催化特性。在人293胚胎肾细胞中过表达的酶通过两个步骤将雄酮转化为表雄酮：将雄酮氧化为5α-雄甾烷-3,17-二酮，然后将后者还原为表雄酮。逆反应，3(β-->α)-羟基类固醇差向异构，大约弱 10 倍。这些结果通过V-max/K-m测定得到证实，这表明该酶比逆反应更有效地催化雄酮氧化为5α-雄甾烷-3,17-二酮和5α-雄甾烷-3,17-二酮还原为表雄酮。在培养的完整转染细胞中也观察到遵循3（α-->β）方向的选择性催化反应，这更好地反映了生理条件。体外测定表明，重组酶更喜欢 NAD(+) 和 NADH 作为辅因子，并且可以识别 C-19 和 C-21 3 α-羟基类固醇作为底物，DNA 序列分析预测蛋白质含有 317 个氨基酸。 RT-PCR的组织分布分析表明，该酶的mRNA在多种组织中表达，包括肝脏、脑、前列腺、肾上腺和子宫，其中在肝脏中表达最丰富。由于活性羟基类固醇通常以立体特异性方式发挥作用，因此 3(α-->β)-HSE 可能在调节各种类固醇的生物活性中发挥重要作用。
In this report, we describe the isolation and characterization of a cDNA encoding an enzyme that exhibits catalytic characteristics of a 3(alpha-->beta)-hydroxysteroid epimerase (3(alpha-->beta)-HSE). The enzyme overexpressed in human 293 embryonic kidney cells transforms androsterone into epi-androsterone in two steps: the oxidation of androsterone to 5 alpha-androstane-3,17-dione, followed by the reduction of the latter to epi-androsterone. The reverse reaction, 3(beta-->alpha)-hydroxysteroid epimeration, is approximately 10-fold weaker. These results are confirmed by V-max/K-m determination, which shows that the enzyme catalyzes the oxidation of androsterone to 5 alpha-androstane-3,17-dione and the reduction of 5 alpha-androstane-3,17-dione to epi-androsterone more efficiently than the reverse reactions. The selective catalysis of the reaction following the 3(alpha-->beta) direction is also observed in intact transfected cells in culture, which better reflect physiological conditions. In vitro assays reveal that the recombinant enzyme prefers NAD(+) and NADH as cofactors and could recognize both C-19 and C-21 3 alpha-hydroxysteroids as substrates, DNA sequence analysis predicts a protein of 317 amino acids. Tissue distribution analysis using RT-PCR reveals that the mRNA of the enzyme is expressed in various tissues, including liver, brain, prostate, adrenal, and uterus, with the most abundant expression in the liver. Because active hydroxysteroids generally exert their effect in a stereospecific manner, 3(alpha-->beta)-HSE could thus potentially play an important role in regulating the biological activities of various steroids.