STRUCTURE/FUNCTION OF 3A-HYDROXYSTEROID DEHYDROGENASE

羟基类固醇脱氢酶（HSD）在人的免疫系统中起关键作用。 所有类固醇激素的生物合成和失活。 在目标 它们通过相互转化来调节核受体的占有率 强效类固醇激素及其同源无活性代谢物。 HSDs 属于两个蛋白质超家族，短链脱氢酶/ 还原酶（SDR）和醛酮还原酶（AKR）。 大鼠肝 3 α-HSD是最彻底表征的HSD，是一种AKR。 脱辅基酶[E]及其二元复合物[E NADP+]的晶体结构 与竞争性抑制剂[E NADP+睾酮]的三元复合物 描述了 3 α-HSD的结构-功能研究将提供 对所有类固醇中催化和配体识别的独特见解 代谢AKR（例如，3alpha-、17 beta-和20 alpha-HSDs，以及delta 4-3酮甾类5 β-还原酶）。 重组大鼠肝3 α-HSD（rr 3 α-HSD）的X射线晶体结构 HSD），含有5 α-二氢睾酮（5 α-DHT）或5 β- 目前，正在寻找DHT。 在这些结构中，3-酮类固醇底物是 平面（A/B trans-ring融合）或显著弯曲（A/B cis- 环融合）并将鉴定催化酸。 的结构 重组人2型3 α-HSD NADP+ 4-雄甾烯-3，17-二酮 也在寻求复杂。 该AKR具有3alpha和17 beta-HSD 活性，可以调节前列腺雄激素受体占有率，但结合 类固醇向后（D环而不是A环第一）和颠倒 （α面在β面方向上）。 使用rr 3 α-HSD，停止- 流动荧光光谱法，初级氘（4 R-NAD（P）D），和 溶剂（D20）动力学同位素效应将决定运动是否 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是反应中的速率限制。 化学步骤 使用催化四分体的突变体的pH速率曲线 (Y55、H117、K84和D50）将揭示一般酸的身份 通过滴定。 参与辅因子结合的残基将突变为 或者反转氢化物转移的立体化学，或者改变 NADPH优先于NADH。 类固醇口袋的知识 利用改变特异性：（1）5 β-还原酶活性将被 通过突变四分体残基引入;（2）20 α-HSD活性将 通过突变类固醇口袋中的残基或通过 构建3 α/20 α-HSD嵌合体，其中 口袋交换;和（4）C-末端环（一个主要的决定因素， 3 α-17 β-和20 α-HSD特异性）将随机 通过噬菌体展示诱变。

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.