Computational Study of PmHMGR Thiohemiacetal Breakdown Transition State

Abstract

3-hydroxyl-3-methylglutaryl CoA reductase (HMGR) catalyzes the rate determining step in the isoprenoid pathway using two NADH molecules. Even though this enzyme is targeted by statins, the atomistic details of the mechanism are still unknown. This objective can be met by combination of computational biophysics and experimental validations through time-resolved Laue crystallography for all steps of the reaction. The catalysis of HMGR can be broken down into three major chemical transformations including two hydride transfers and a thiohemiacetal breakdown step. Moreover, since two cofactor molecules are required, a cofactor exchange process also occurs after the first hydride transfer and before the second hydride transfer. Thiohemiacetal is a known intermediate resulting from the first hydride transfer step. However, the product of the thiohemiacetal breakdown step hypothesized to be mevaldehyde, has not been experimentally validated. This theoretical study explores the atomic details of the enzyme and ligands for the thiohemiacetal breakdown step using the Pseudomonas mevalonii HMGR. Theozymes and QM/MM ONIOM models were used to identify the transition state were performed to reveal the catalytic residues. These models have revealed that the Nδ of His381 is involved in stabilizing the negative charge in the thiolate anion. Ser85 forms a hydrogen bonding network with the first amide of thiohemiacetal ligand, a function performed by His381 in the preceding step first hydride transfer. Transition state force fields (TSFF) using the quantum guided molecular mechanics (Q2MM) program will then be used to perform µsec timescale molecular dynamics simulations. This project focuses on not only developing computational biophysics methods for studying enzyme mechanisms but also uses time-resolved x-ray crystallography to obtain snapshots along the HMGR reaction pathway which involves multiple transformations of the ligand in the same active site, a large flap domain movement, and a cofactor exchange.