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

Abstract

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.

Figure 1.

(a) PmHMGR crystal structure (PDB: 4i4b) showing two monomers (chain A in purple), flap domain in pink ribbon, and ligands in ball–stick models (NAD in cyan and dithiohemiacetal in yellow). (b) PmHMGR catalytic site showing ligands and conserved residues. (c) The mevaldyl-CoA decomposition reaction showing Glu83 as the proton acceptor and His381 as the proton donor.

Figure 2.

Theozyme model for PmHMGR thiohemiacetal decomposition highlighting the frozen carbon atoms (pink) which mimic attachment to the rest of the protein. Other colors: gray = C, white = H, blue = N, red = O, and yellow = S.

Figure 3.

(a) QM/MM model composed of the 8 Å core (gray surface model) of 4i4b measured from the ligands and residues (ball and stick models). (b) QM layer with 106 atoms highlighting the atoms (pink) at the QM/MM partition.

Figure 4.

Reactant, transition state, and product geometries for mevaldyl-CoA decomposition calculated at M06/6–31G** showing the changes in bond lengths.

Figure 5.

Overlay of the TS geometries from the theozyme and QM/MM models.

Figure 6.

Activation and reaction energies (SCF single point calculations, relative to reactant, in kcal/mol) for the theozyme and QM layer (structures illustrated in Figures 4 and S4).