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HMG-CoA reductase catalyzes the four-electron reduction of HMG-CoA to mevalonate and is an enzyme of considerable biomedical relevance because of the impact of its statin inhibitors on public health. Although the reaction has been studied extensively using X-ray crystallography, there are surprisingly no computational studies that test the mechanistic hypotheses suggested for this complex reaction. Theozyme and quantum mechanical (QM)/molecular mechanical (MM) calculations up to the B3LYP/6-31g(d,p)//B3LYP/6-311++g(2d,2p) level of theory were employed to generate an atomistic description of the enzymatic reaction process and its energy profile. The models generated here predict that the catalytically important Glu83 is protonated prior to hydride transfer and that it acts as the general acid or base in the reaction. With Glu83 protonated, the activation energies calculated for the sequential hydride transfer reactions, 21.8 and 19.3 kcal/mol, are in qualitative agreement with the experimentally determined rate constant for the entire reaction (1 s(-1) to 1 min(-1)). When Glu83 is not protonated, the first hydride transfer reaction is predicted to be disfavored by >20 kcal/mol, and the activation energy is predicted to be higher by >10 kcal/mol. While not involved in the reaction as an acid or base, Lys267 is critical for stabilization of the transition state in forming an oxyanion hole with the protonated Glu83. Molecular dynamics simulations and MM/Poisson-Boltzmann surface area free energy calculations predict that the enzyme active site stabilizes the hemithioacetal intermediate better than the aldehyde intermediate. This suggests a mechanism in which cofactor exchange occurs before the breakdown of the hemithioacetal. Slowing the conversion to aldehyde would provide the enzyme with a mechanism to protect it from solvent and explain why the free aldehyde is not observed experimentally. Our results support the hypothesis that the pK(a) of an active site acidic group is modulated by the redox state of the cofactor. The oxidized cofactor and deprotonated Glu83 are closer in space after hydride transfer, indicating that indeed the cofactor may influence the pK(a) of Glu83 through an electrostatic interaction. The enzyme is able to catalyze the transfer of a hydride to the structurally and electronically distinct substrates by maintaining the general shape of the active site and adjusting the electrostatic environment through acid-base chemistry. Our results are in good agreement with the well-studied hydride transfer reactions catalyzed by liver alcohol dehydrogenase in calculated energy profile and reaction geometries despite different mechanistic functionalities.

Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg(2+)-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g., Staphylococcus, Streptococcus, and Enterococcus spp.), and thus the enzymes of the mevalonate pathway are ideal antimicrobial targets. MDD belongs to the GHMP superfamily of metabolite kinases that have been extensively studied for the past 50 years, yet the crystallization of GHMP kinase ternary complexes has proven to be difficult. To further our understanding of the catalytic mechanism of GHMP kinases with the purpose of developing broad spectrum antimicrobial agents that target the substrate and nucleotide binding sites, we report the crystal structures of wild-type and mutant (S192A and D283A) ternary complexes of Staphylococcus epidermidis MDD. Comparison of apo, MVAPP-bound, and ternary complex wild-type MDD provides structural information about the mode of substrate binding and the catalytic mechanism. Structural characterization of ternary complexes of catalytically deficient MDD S192A and D283A (k(cat) decreased 10(3)- and 10(5)-fold, respectively) provides insight into MDD function. The carboxylate side chain of invariant Asp(283) functions as a catalytic base and is essential for the proper orientation of the MVAPP C3-hydroxyl group within the active site funnel. Several MDD amino acids within the conserved phosphate binding loop ("P-loop") provide key interactions, stabilizing the nucleotide triphosphoryl moiety. The crystal structures presented here provide a useful foundation for structure-based drug design.

The sequences and three-dimensional structures of the galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase (GHMP) family were compared to identify highly conserved surface residues. The functions of these solvent-accessible residues were assessed by determining the effects of their substitution, via mutagenesis, on the initial-rate parameters of a representative member of the GHMP kinase family, phosphomevalonate kinase from Streptococcus pneumoniae. What emerges from this study is a profile of the conserved surface-linked functions of the family. Certain substitutions produce highly selective effects on the steady-state affinity of a particular substrate, while one residue, Asp150, appears to be a pure k(cat) effector. Substitutions elsewhere affect multiple initial-rate parameters with varying, and sometimes compensatory, patterns. An alpha-helix that repositions during catalysis was substituted along its length to assess how its different segments contribute to catalysis-the substrate-proximal edge of the helix affects ATP recognition and k(cat), while the distal edge affects recognition of both substrates without affecting turnover. GHMP kinase mutations at the conserved surface residues corresponding to Ser291 and Ala293 in phosphomevalonate kinase are linked to mevalonic acid deficiency, which can lead to early fatality, and galactokinase deficiency, which causes cataracts. Our results suggest that the molecular basis for this particular galactokinase deficiency is an increase in the K(m) for galactose.