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

人类固醇 5β-还原酶 (AKR1D1) 的动力学和结构分析

• 批准号: 8127256
• 负责人: Mo Chen
• 金额: $ 5.13万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8127256
• 关键词: Address; Affect; Androgens; Behavior; Bile Acid Biosynthesis Pathway; Bile Acids; Binding; Catalysis; Characteristics; Chemicals; Cholestasis; Crystallization; Depressed mood; Detergents; Disease; Enzymes; Family; Fat-Soluble Vitamin; Fatty acid glycerol esters; Glucocorticoids; Grant; Human; In Vitro; Investigation; Isotopes; Ketosteroids; Kinetics; Knowledge; Ligands; Measurement; Molecular; Mutation; NADP; Nuclear Receptors; Outcome Study; Oxidoreductase; Point Mutation; Process; Progestins; Reaction; Research; Roentgen Rays; Role; Sex Functioning; Solubility; Solvents; Steroids; Structure; base; chemical kinetics; cofactor; enzyme activity; improved; inhibitor/antagonist; insight; mutant; neonatal hepatitis; pi bond; reproductive function; steroid hormone; steroid hormone metabolism

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.

描述（申请人提供）：人类固醇52还原酶（醛酮还原酶1D1，AKR1D1）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-还原使胆汁酸具有类似洗涤剂的特性，可以乳化脂肪和脂溶性维生素。 AKR1D1 还可使雄激素、孕激素和糖皮质激素失活，并启动类固醇激素清除。 AKR1D1 缺乏会诱发新生儿肝炎和胆汁淤积，这可能是致命的。 AKR1D1 在性和生殖功能中的确切作用仍在研究中。 AKR1D1 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 AKR1D1 的动力学和化学机制，并研究自然突变 P133R 导致疾病的分子基础。该项目的第一个目标是阐明 AKR1D1 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 52 还原中涉及的速率决定步骤。将利用初级和溶剂动力学同位素效应来解决双键还原的化学机制。第二个目标将集中于 P133R 突变体的动力学分析和 X 射线晶体结构测定。提出了一种缓慢的类固醇产品释放过程来抑制 P133R 突变体活性。将阐明 P133R 突变体的宏观速率常数，并将其与野生型 AKR1D1 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 的环中的结构变化，这可能会影响底物结合和催化。 公共健康相关性：该项目旨在了解人体必需的类固醇转化酶 52-还原酶 (AKR1D1) 的机制和结构。野生型酶和与疾病相关的突变体 P133R 都将受到研究。从该研究中获得的信息将为醛酮还原酶 (AKR) 催化和 AKR1D1 相关胆汁酸缺乏提供新的见解。