STRUCTURE & FUNCTION OF 3A-HYDROXYSTEROID DEHYDROGENASE

哺乳动物羟基类固醇脱氢酶（HSDs）在哺乳动物的代谢中起关键作用。 类固醇激素的生物合成和失活。 分子克隆 表明HSDs属于至少两个不同的蛋白质家族， 短链醇脱氢酶和醛酮还原酶（AKR） 超级家庭 大鼠肝脏3 α-HSD（EC 1.1.1.50）是AKR的成员 家庭和灭活循环雄激素，孕激素和 糖皮质激素 3 α-HSD的cDNA已被克隆、测序， 在E.杆菌 脱辅基酶的晶体结构已被 以近原子分辨率（3.0 A）解决，代表了第一个3- 哺乳动物HSD的三维结构。 3alpha-HSD结构采用 a（beta/alpha）8桶基序（磷酸丙糖异构酶桶）和缺乏 a结合NAD的罗斯曼折叠（P）（H）。 要将结构与功能联系起来， 计划进行以下研究。 [E.NADPH]二元和三元配合物的X射线晶体结构 [E.NADPH.类固醇]的3 α-HSD现在寻求原子分辨率。 的 现有的3.0 A晶体结构暗示： 氧化还原（Tyr 55、Lys 84和Asp 50）;结合吡啶中的残基 延伸反构象的核苷酸（Tyr 216，Asn 167，Ser 166，Arg 270 和Arg 276）;和用于结合类固醇的椭圆形疏水口袋 激素（Tyr 55、Trp 86、Phe 118、Phe 128、Phe 129和Trp 227）。 这些残基 会发生变异 特定地点效应将与全球效应区分开来。 通过比较r3 α-HSD的动力学指纹和光谱指纹 突变蛋白质。 动力学参数（Km、kcat、kcat/Km和 pH-kcat曲线）和配体亲和力（辅因子或类固醇）将决定 哪些氨基酸在催化作用中很重要， 配体结合 验证NADPH与3 α-HSD结合的原子模型 该位点将用腺苷进行亲和标记[例如，3 '-和5'-p- （氟磺酰基）-苯甲酰基-腺苷]和烟酰胺[例如，卤乙酰基，卤乙酰基 吡啶]衍生物和修饰的氨基酸。 开环甾 机制基础灭活剂将用于标记参与的氨基酸， 类固醇识别 这些开环甾体缺乏A和D环，但含有 炔醇，其当量为C-3 [1，10-seco-5alpha- 雌甾-1-炔-3（R，S）-17 β-二醇]或C-17 [17（r，S）-羟基-14，15-开环雌甾-4-醇]。 en-16-yn-3-one]位置。 这两种醇都被3alpha-HSD氧化 与共价修饰酶的相应炔属酮反应。 由于氧化在C-3和C-17位置都被催化， 这些部分类固醇必须反向进入活性部位。 以识别 由炔酮标记的氨基酸，放射性标记灭活 将对酶进行肽图谱和测序。 我们的研究 将提供结构基础，通过该结构基础，HSD识别它们的配体， 类固醇激素，并可能适用于所有属于 AKR超级大家庭

Mammalian hydroxysteroid dehydrogenases (HSDs) play pivotal roles in the biosynthesis and inactivation of steroid hormones. Molecular cloning indicates that HSDs belong to at least two distinct protein families the short-chain alcohol dehydrogenases, and the aldo-keto reductase (AKR) super-family. Rat liver 3alpha-HSD(EC 1.1.1.50) is a member of the AKR family and inactivates circulating androgens, progestins and glucocorticoids. The cDNA for 3alpha-HSD has been cloned, sequenced and over-expressed in E. coli. The crystal structure of the apoenzyme has been solved at near atomic resolution (3.0 A) and represents the first 3- dimensional structure of a mammalian HSD. The 3alpha-HSD structure adopts a (beta/alpha)8 barrel motif (triosephosphate isomerase barrel) and lacks a Rossmann fold for binding NAD(P)(H). To relate structure to function the following studies are planned. X-ray crystallographic structures of binary [E.NADPH] and ternary complexes [E.NADPH.Steroid] of 3alpha-HSD are now sought at atomic resolution. The existing 3.0 A crystal structure implicates: a catalytic triad in oxidoreduction (Tyr55, Lys84, and Asp50); residues in binding pyridine nucleotide in an extended anti-conformation (Tyr216, Asn167, Ser166, Arg270 and Arg276); and an elliptical hydrophobic pocket for binding steroid hormone (Tyr55, Trp86, Phe118 Phe128, Phe129 and Trp227). These residues will be mutated. Site-specific effects will be distinguished from global effects by comparing the kinetic and spectral fingerprints of r3alpha-HSD with mutant proteins. Changes in kinetic parameters (Km, kcat, kcat/Km and pH-kcat profiles), and ligand affinity (cofactor or steroid) will determine which amino acids are important in catalysis and which are important in ligand binding. To verify atomic models for binding NADPH to 3alpha-HSD this site will be affinity-labeled with adenosine [e.g., 3'- and 5'-p- (fluorosulfonyl)-benzoyl-adenosine] and nicotinamide [e.g., haloacetyl- pyridine] derivatives and modified amino acids identified. Secosteroid mechanism-base inactivators will be used to label amino acids involved in steroid recognition. These secosteroids lack A and D rings but contain acetylenic alcohols at the equivalent of either the C-3 [1,10-seco-5alpha- estr-1-yne-3(R,S)-17beta-diol] or C-17 [17(r,S)-hydroxy-14,15-secoestr-4- en-16-yn-3-one] positions. Both these alcohols are oxidized by 3alpha-HSD to the corresponding acetylenic ketones which covalently modify the enzyme. Since oxidation is catalyzed at both the C-3 and C-17 positions one of these partial steroids must enter the active site backwards. To identify amino acids tagged by the acetylenic ketones, radiolabeled inactivated enzyme will be subjected to peptide mapping and sequencing. Our studies will provide a structural basis by which HSDs recognize their ligands and inactivate steroid hormones and may be applicable to all HSDs that belong to the AKR super-family.