Time-Resolved Crystallography Measurements Elucidating the Mechanism of Bacterial HMG-CoA Reductase

Purohit, Vatsal; Steussy, C.N.; Schmidt, Timothy R.; Critchelow, Chandra J.; Rosales, Tony; Stauffacher, Cynthia V.; Helquist, Paul; Weist, Olaf

doi:10.1016/j.bpj.2018.11.131

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“…Time-dependent crystallographic experiments also lack electron densities for conserved water molecules, show different orientations for catalytic residues, and show formation of C−S bond after a hydride transfer to mevalonate. 63 Similar to other enzymes with thiohemiacetal intermediates, 9,10,12,13 PmHMGR has cysteine residues, but unlike these enzymes, PmHMGR's cysteine residues (Cys156 and Cys296) showed minimal effect on the enzyme activity when mutated to alanine. 38 This verifies that PmHMGR thiohemiacetal decomposition is not facilitated by these cysteines.…”

Section: ■ Discussionmentioning

confidence: 97%

“…Mevaldehyde has been used to run half-reactions (one reduction in HMGRs) to give mevalonate, but extensive experimental studies have indicated that this intermediate does not leave the active site. ,,, While it is possible for this thiohemiacetal to be hydrolyzed by a water molecule, the subsequent reaction does not favor it, thereby making this a less favorable decomposition mechanism. Time-dependent crystallographic experiments also lack electron densities for conserved water molecules, show different orientations for catalytic residues, and show formation of C–S bond after a hydride transfer to mevalonate …”

Section: Discussionmentioning

confidence: 99%

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Computational Study of Base-Catalyzed Thiohemiacetal Decomposition in Pseudomonas mevalonii HMG-CoA Reductase

et al. 2023

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Thiohemiacetals are key intermediates in the active sites of many enzymes catalyzing a variety of reactions. In the case of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A reductase (PmHMGR), this intermediate connects the two hydride transfer steps where a thiohemiacetal is the product of the first hydride transfer and its breakdown forms the substrate of the second one, serving as the intermediate during cofactor exchange. Despite the many examples of thiohemiacetals in a variety of enzymatic reactions, there are few studies that detail their reactivity. Here, we present computational studies on the decomposition of the thiohemiacetal intermediate in PmHMGR using both QM-cluster and QM/MM models. This reaction mechanism involves a proton transfer from the substrate hydroxyl to an anionic Glu83 followed by a C−S bond elongation stabilized by a cationic His381. The reaction provides insight into the varying roles of the residues in the active site that favor this multistep mechanism.

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