STRUCTURE/FUNCTION OF 3A-HYDROXYSTEROID DEHYDROGENASE

羟基类固醇脱氢酶(HSD)在细胞周期中起着关键作用。 所有类固醇激素的生物合成和失活。在目标中 它们通过相互转换来调节核受体的占用 强效类固醇激素及其同源非活性代谢物。HSD 属于两个蛋白质超家族：短链脱氢酶/ 还原酶(SDRs)和醛酮还原酶(AKRs)。大鼠肝脏 3α-HSD是AKR中特征性最强的HSD。 脱辅酶[E]及其二元络合物[E-NADP+]的晶体结构 和带有竞争性抑制剂的三元络合物[E-NADP+睾酮] 都被描述了。对3α-HSD的结构-功能研究将提供 对所有类固醇的催化和配体识别有独到的见解 代谢AKRs(例如，3α、17β和20α-HSD和Delta 4-3酮类固醇5β-还原酶)。 重组大鼠肝脏3α-HSD(rr3α-HSD)的X射线晶体结构 HSD)含有5α-双氢睾酮(5α-DHT)或5β-二氢睾酮(5α-DHT)。 现在正在寻找DHT。在这些结构中，3-酮类固醇底物是 平面(A/B跨环融合)或显著弯曲(A/B顺式- 环融合)，并将鉴定催化酸。的结构 重组人23Alpha-HSD NADP+4-雄烯-3，17-二酮 综合体也受到追捧。这款AKR有3α和17β-HSD 活性，可能调节前列腺雄激素受体的占位，但结合 类固醇倒置(首先是D环而不是A环)和颠倒 (Alpha-Face在beta-Face方向)。使用rr3pha-HSD，停止- 流动荧光光谱分析，一次氢(4R-NAD(P)D)，和 溶剂(D20)的动力学同位素效应将决定运动是否 核苷酸钳制环在动力学机制中是限速的， 以及氢化物转移或质子捐赠是否在 化学步骤。利用催化四分体突变体的pH速率谱 (Y55、H117、K84和D50)将揭示总酸的特征 通过滴定。参与辅因子结合的残基将突变为 要么颠倒氢化物转移的立体化学，要么改变 偏好从NADPH到NADH。对类固醇口袋的了解将是 利用来改变特异性：(1)5β-还原酶活性将 由突变的四分体残基引入；(2)20α-HSD活性将 通过类固醇口袋中的突变残基或通过 构建3α/20α-HSD嵌合体，在该嵌合体中 口袋被交换；以及(4)C-端环(主要决定因素 3α-17β-和20α-HSD特异性)将是随机的 噬菌体展示诱变。

Hydroxysteroid dehydrogenases (HSDs) play pivotal roles in the biosynthesis and inactivation of all steroid hormones. In target tissues they regulate occupancy of nuclear receptors by interconverting potent steroid hormones with their cognate inactive metabolites. HSDs belong to two protein superfamilies the short-chain dehydrogenase/ reductases (SDRs) and the aldo-keto reductases (AKRs). Rat liver 3alpha-HSD is the most thoroughly characterized HSD that is an AKR. Crystal structures of the apoenzyme [E], its binary complex [E NADP+] and ternary complex with a competitive inhibitor [E NADP+ testosterone] are described. Structure-function studies on 3alpha-HSD will provide unique insight into catalysis and ligand recognition in all steroid metabolizing AKRs (e.g., 3alpha-, 17beta-, and 20alpha-HSDs, and delta 4-3 ketosteriod 5beta-reductase). X-ray crystal structures of recombinant rat liver 3alpha-HSD (rr3alpha- HSD) containing either 5alpha-dihydrotestosterone (5alpha-DHT) or 5beta- DHT are now sought. In these structures the 3-ketosteroid substrate is either planar (A/B trans-ring fusion) or significantly bent (A/B cis- ring fusion) and will identify the catalytic acid. The structure of recombinant human type 2 3alpha-HSD NADP+ 4-androstene-3, 17-dione complex is also sought. This AKR has both 3alpha- and 17beta-HSD activity, may regulate prostate androgen receptor occupancy, yet binds steroids backwards (D ring instead of A ring first) and upside down (alpha-face in the beta-face orientation). Using rr3alpha-HSD, stopped- flow fluorescence spectroscopy, primary deuterium (4R-NAD(P)D), and solvent (D20) kinetic isotope effects will determine whether movement of a nucleotide-clamping loop is rate-limiting in the kinetic mechanism, and whether hydride transfer or proton donation is rate-limiting in the chemical step. pH rate profiles using mutants of the catalytic tetrad (Y55, H117, K84 and D50) will reveal the identity of the general acid by titration. Residues involved in cofactor binding will be mutated to either invert the stereochemistry of hydride transfer or change preference from NADPH to NADH. Knowledge of the steroid pocket will be exploited to alter specificity: (1) 5beta-reductase activity will be introduced by mutating tetrad residues; (2) 20alpha-HSD activity will be engineered by either mutating residues in the steroid pocket or by constructing 3alpha/20alpha-HSD chimeras in which loop regions of the pocket are swapped; and (4) the C-terminal loop (a major determinant of 3alpha- 17beta- and 20alpha-HSD specificity) will be randomly mutagenized by phage-display.