Kinetic and structural analysis of human steroid 5beta-reductase (AKR1D1)

描述（由申请人提供）：人类固醇-52-还原酶（醛酮还原酶1D 1，AKR 1D 1）是胆汁酸生物合成和类固醇激素代谢中最关键的酶之一。52-还原赠款胆汁酸具有类似洗涤剂的特性，以去除脂肪和脂溶性维生素。AKR 1D 1还使雄激素、孕激素和糖皮质激素失活，并启动类固醇激素清除。AKR 1D 1的缺乏会导致新生儿肝炎和胆汁淤积，这可能是致命的。AKR 1D 1在性和生殖功能中的确切作用仍在研究中。AKR 1D 1催化类固醇-52-还原的机制尚未得到严格阐明。该提案的目的是建立AKR 1D 1的动力学和化学机制，并研究自然突变P133 R导致疾病的分子基础。在本项目的第一个目标中，将阐明AKR 1D 1的完整动力学机制。将采用稳态和瞬态动力学测量的组合来确定宏观速率常数，并确定参与52-还原的速率决定步骤。主要和溶剂动力学同位素效应将被用来解决双键还原的化学机制。第二个目标将集中在动力学分析和X-射线晶体结构测定的P133 R突变体。提出了一种缓慢的类固醇产物释放过程来抑制P133 R突变体活性。将阐明P133 R突变体的宏观速率常数，并与野生型AKR 1D 1的宏观速率常数进行比较，以验证该假设。突变体的晶体结构将阐明含有残基P133的环中可能影响底物结合和催化的结构变化。 公共卫生相关性：该项目旨在了解人体必需的类固醇转化酶52-还原酶（AKR 1D 1）的机制和结构。野生型酶和疾病相关突变体P133 R都将被研究。从这项研究中获得的信息将为醛酮还原酶（AKR）催化和AKR 1D 1相关的胆汁酸缺乏症提供新的见解。

DESCRIPTION (provided by applicant): Human steroid-52-reductase (aldo-keto reductase 1D1, AKR1D1) is one of the most critical enzymes in bile acid biosynthesis and steroid hormone metabolism. 52-Reduction grants bile acids the detergent-like characteristics to emulsify fats and fat soluble vitamins. AKR1D1 also inactivates androgens, progestins and glucocorticoids and initiates steroid hormone clearance. Deficiency in AKR1D1 induces neonatal hepatitis and cholestasis that can be fatal. The exact role of AKR1D1 in sexual and reproductive functions is still under investigation. The mechanism of steroid-52-reduction catalyzed by AKR1D1 has not been rigorously elucidated. The objective of this proposal is to establish the kinetic and chemical mechanisms for AKR1D1 and investigate the molecular basis by which a natural mutation P133R leads to disease. In the first aim of this project, the complete kinetic mechanism for AKR1D1 will be elucidated. A combination of steady-state and transient-state kinetic measurements will be employed to determine macroscopic rate constants and identify the rate-determining step involved in 52-reduction. Primary and solvent kinetic isotope effects will be utilized to address the chemical mechanism of the double bond reduction. The second aim will focus on the kinetic analysis and X-ray crystal structure determination of the P133R mutant. A slow steroid product release process was proposed to depress P133R mutant activity. The macroscopic rate constants of the P133R mutant will be elucidated and compared to that of the wild type AKR1D1 to validate the hypothesis. The crystal structure of the mutant will illuminate structural changes in the loop containing residue P133 that may affect substrate binding and catalysis. PUBLIC HEALTH RELEVANCE: This project aims to understand the mechanism and structure of an essential human steroid transforming enzyme, 52-reductase (AKR1D1). Both the wild type enzyme and a disease-related mutant, P133R, will be investigated. The information obtained from the study will provide new insights into aldo-keto reductase (AKR) catalysis and AKR1D1-associated bile acid deficiency.