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

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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.