3-hydroxyl-3-methylglutaryl CoA reductase (HMGR) catalyzes the rate-determining step in the isoprenoid pathway using two equivalents of NADH. Despite significant biomedical interest in the bacterial HMGR and its human homolog, many atomistic details of the mechanism are still unknown. We use a combination of computational studies using DFT and transition state force field (TSFF) calculations and time-resolved crystallography triggered through pH-jump. The catalysis of HMGR can be divided into three major chemical transformations including two hydride transfers separated by a base-catalyzed thiohemiacetal decomposition. This theoretical study explores the atomic details of the enzyme and ligands for the thiohemiacetal breakdown in PmHMGR whose catalytic site is structurally similar to that of human HMGR. Theozymes models are used to calculate the transition state of thiohemiacetal decomposition. Transition states calculated at the M06/6-31G(d,p) level indicate a higher energy of activation for the theozyme models containing NAD relative to the NADH ones. They reveal that the Nδ of His381 stabilizes the negative charge of the thiolate anion. While QM/MM models show a smaller C-S bond elongation relative to the theozyme models, the former models establish Ser85 hydrogen-bonding network with substrate amide, a function performed by His381 in the first hydride transfer. To elucidate the role of His381 in its different protonation states, molecular dynamics simulations reveal a greater stabilization of the active site with a positively charged His381. The theozyme models are used as reference data for generating TSFFs using the quantum guided molecular mechanics (Q2MM) program for the thiohemiacetal decomposition. The TSFF will then be used to perform µsec timescale molecular dynamics simulations to probe further mechanistic questions such as the flap domain movement that facilitates the cofactor exchange. This project focuses on not only developing computational biophysics methods for studying enzyme mechanisms but also using 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.

Computational study of base-catalyzed thiohemiacetal decomposition in Pseudomonas mevalonii HMG-CoA reductase

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