Point Mutations In Candida Glabrata 3-Hydroxy-3-Methyl Glutaryl Coenzyme A Reductase (HMGRCg) An Antifungal Target, Decrease Enzymatic Activity And Substrate/Inhibitor Affinity

Abstract

An alternative target for antifungal drugs is 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR), a key enzyme in the ergosterol biosynthesis pathway. The aim of this study was to obtain, purify, characterize, and overexpress five point mutations in highly conserved regions of the catalytic domain of Candida glabrata HMGR (HMGRCg) to explore the function of key amino acid residues. Glutamic acid (Glu) was substituted by glutamine in the E680Q mutant (at the dimerization site), Glu by glutamine in E711Q (at the substrate binding site), aspartic acid by alanine in D805A and methionine by arginine in M807R (the latter two at the cofactor binding site). A double mutation, E680Q-M807R, was also made. The in vitro enzymatic activity decreased significantly in all recombinant (versus wild-type) HMGRCg, and the in silico binding energy for simvastatin, alpha-asarone and the substrate HMG-CoA was also lower for the mutants. The lowest enzymatic activity and binding energy was displayed by E711Q, suggesting that Glu711 (in the substrate binding site) is an important residue for enzymatic activity. The double mutant HMGRCg E680Q-M807R exhibited the second lowest enzymatic activity. The current findings provide insights into the role of residues in the catalytic site of HMGRCg.