Active site binding modes of inhibitors of Staphylococcus aureus mevalonate diphosphate decarboxylase from docking and molecular dynamics simulations.

Bacterial mevalonate diphosphate decarboxylase (MDD) is an attractive therapeutic target for antibacterial drug development. In this work, we discuss a combined docking and molecular dynamics strategy toward inhibitor binding to bacterial MDD. The docking parameters utilized in this study were first validated with observations for the inhibitors 6-fluoromevalonate diphosphate (FMVAPP) and diphosphoglycolylproline (DPGP) using existing structures for the Staphylococcus epidermidis enzyme.

Metabolic Rewiring by Oncogenic BRAF V600E Links Ketogenesis Pathway to BRAF-MEK1 Signaling.

Many human cancers share similar metabolic alterations, including the Warburg effect. However, it remains unclear whether oncogene-specific metabolic alterations are required for tumor development. Here we demonstrate a "synthetic lethal" interaction between oncogenic BRAF V600E and a ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL).

Role of VMH ketone bodies in adjusting caloric intake to increased dietary fat content in DIO and DR rats.

The objective of this study was to determine the potential role of astrocyte-derived ketone bodies in regulating the early changes in caloric intake of diet induced-obese (DIO) versus diet-resistant (DR) rats fed a 31.5% fat high-energy (HE) diet. After 3 days on chow or HE diet, DR and DIO rats were assessed for their ventromedial hypothalamic (VMH) ketone bodies levels and neuronal ventromedial hypothalamic nucleus (VMN) sensing using microdialysis coupled to continuous food intake monitoring and calcium imaging in dissociated neurons, respectively.

Inhibition of bacterial mevalonate diphosphate decarboxylase by eriochrome compounds.

Mevalonate diphosphate decarboxylase (MDD; EC 4.1.1.

Identification in Haloferax volcanii of phosphomevalonate decarboxylase and isopentenyl phosphate kinase as catalysts of the terminal enzyme reactions in an archaeal alternate mevalonate pathway.

Mevalonate (MVA) metabolism provides the isoprenoids used in archaeal lipid biosynthesis. In synthesis of isopentenyl diphosphate, the classical MVA pathway involves decarboxylation of mevalonate diphosphate, while an alternate pathway has been proposed to involve decarboxylation of mevalonate monophosphate. To identify the enzymes responsible for metabolism of mevalonate 5-phosphate to isopentenyl diphosphate in Haloferax volcanii, two open reading frames (HVO_2762 and HVO_1412) were selected for expression and characterization.

Expression in Haloferax volcanii of 3-hydroxy-3-methylglutaryl coenzyme A synthase facilitates isolation and characterization of the active form of a key enzyme required for polyisoprenoid cell membrane biosynthesis in halophilic archaea.

Enzymes of the isoprenoid biosynthetic pathway in halophilic archaea remain poorly characterized, and parts of the pathway remain cryptic. This situation may be explained, in part, by the difficulty of expressing active, functional recombinant forms of these enzymes. The use of newly available expression plasmids and hosts has allowed the expression and isolation of catalytically active Haloferax volcanii 3-hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (EC 2.

Molecular docking and NMR binding studies to identify novel inhibitors of human phosphomevalonate kinase.

Phosphomevalonate kinase (PMK) phosphorylates mevalonate-5-phosphate (M5P) in the mevalonate pathway, which is the sole source of isoprenoids and steroids in humans. We have identified new PMK inhibitors with virtual screening, using autodock. Promising hits were verified and their affinity measured using NMR-based (1)H-(15)N heteronuclear single quantum coherence (HSQC) chemical shift perturbation and fluorescence titrations.

Identification and characterization of an extramitochondrial human 3-hydroxy-3-methylglutaryl-CoA lyase.

3-Hydroxy-3-methylglutaryl-CoA lyase-like protein (HMGCLL1) has been annotated in the Mammalian Genome Collection as a previously unidentified human HMG-CoA lyase (HMGCL). To test the validity of this annotation and evaluate the physiological role of the protein, plasmids were constructed for protein expression in Escherichia coli and Pichia pastoris. Protein expression in E.

Structural basis for nucleotide binding and reaction catalysis in mevalonate diphosphate decarboxylase.

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.

Biochemical and structural basis for inhibition of Enterococcus faecalis hydroxymethylglutaryl-CoA synthase, mvaS, by hymeglusin.

Hymeglusin (1233A, F244, L-659-699) is established as a specific β-lactone inhibitor of eukaryotic hydroxymethylglutaryl-CoA synthase (HMGCS). Inhibition results from formation of a thioester adduct to the active site cysteine. In contrast, the effects of hymeglusin on bacterial HMG-CoA synthase, mvaS, have been minimally characterized.

ChemVassa: A New Method for Identifying Small Molecule Hits in Drug Discovery.

ChemVassa, a new chemical structure search technology, was developed to allow rapid in silico screening of compounds for hit and hit-to-lead identification in drug development. It functions by using a novel type of molecular descriptor that examines, in part, the structure of the small molecule undergoing analysis, yielding its "information signature." This descriptor takes into account the atoms, bonds, and their positions in 3-dimensional space.

Ceestatin, a novel small molecule inhibitor of hepatitis C virus replication, inhibits 3-hydroxy-3-methylglutaryl-coenzyme A synthase.

Background: Hepatitis C virus (HCV) chronically infects >170 million persons worldwide and is a leading cause of cirrhosis and hepatocellular carcinoma. The identification of more effective and better-tolerated agents for treating HCV is a high priority. We have reported elsewhere the discovery of the anti-HCV compound ceestatin using a high-throughput screen of a small molecule library.

Crystal structures of Staphylococcus epidermidis mevalonate diphosphate decarboxylase bound to inhibitory analogs reveal new insight into substrate binding and catalysis.

The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in gram-positive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents.

Influence of multiple cysteines on human 3-hydroxy-3-methylglutaryl-CoA lyase activity and formation of inter-subunit adducts.

Human 3-hydroxy-3-methylglutaryl-CoA lyase catalyzes formation of acetyl-CoA and acetoacetate in a reaction that requires divalent cation and is stimulated by sulfhydryl protective reagents. The enzyme is a homodimer and inter-subunit adducts form in the absence of reducing agents or upon treatment with cysteine selective crosslinking agents. To address the influence of cysteines on enzyme activity and formation of inter-subunit and intra-subunit adducts, single serine substitutions have been engineered for each enzyme cysteine.

Enzymes of the mevalonate pathway of isoprenoid biosynthesis.

The mevalonate pathway accounts for conversion of acetyl-CoA to isopentenyl 5-diphosphate, the versatile precursor of polyisoprenoid metabolites and natural products. The pathway functions in most eukaryotes, archaea, and some eubacteria. Only recently has much of the functional and structural basis for this metabolism been reported.

Functional insights into human HMG-CoA lyase from structures of Acyl-CoA-containing ternary complexes.

HMG-CoA lyase (HMGCL) is crucial to ketogenesis, and inherited human mutations are potentially lethal. Detailed understanding of the HMGCL reaction mechanism and the molecular basis for correlating human mutations with enzyme deficiency have been limited by the lack of structural information for enzyme liganded to an acyl-CoA substrate or inhibitor. Crystal structures of ternary complexes of WT HMGCL with the competitive inhibitor 3-hydroxyglutaryl-CoA and of the catalytically deficient HMGCL R41M mutant with substrate HMG-CoA have been determined to 2.

NMR dynamics investigation of ligand-induced changes of main and side-chain arginine N-H's in human phosphomevalonate kinase.

Phosphomevalonate kinase (PMK) catalyzes phosphoryl transfer from adenosine triphosphate (ATP) to mevalonate 5-phosphate (M5P) on the pathway for synthesizing cholesterol and other isoprenoids. To permit this reaction, its substrates must be brought proximal, which would result in a significant and repulsive buildup of negative charge. To facilitate this difficult task, PMK contains 17 arginines and eight lysines.

A visible wavelength spectrophotometric assay suitable for high-throughput screening of 3-hydroxy-3-methylglutaryl-CoA synthase.

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase catalyzes the first physiologically irreversible step in biosynthesis of isoprenoids and sterols from acetyl-CoA. Inhibition of enzyme activity by beta-lactone-containing natural products correlates with substantial diminution of sterol synthesis, identifying HMG-CoA synthase as a potential drug target and suggesting that identification of effective inhibitors would be valuable. A visible wavelength spectrophotometric assay for HMG-CoA synthase has been developed.

Human mevalonate diphosphate decarboxylase: characterization, investigation of the mevalonate diphosphate binding site, and crystal structure.

Expression in Escherichia coli of his-tagged human mevalonate diphosphate decarboxylase (hMDD) has expedited enzyme isolation, characterization, functional investigation of the mevalonate diphosphate binding site, and crystal structure determination (2.4A resolution). hMDD exhibits V(max)=6.

Substrate induced structural and dynamics changes in human phosphomevalonate kinase and implications for mechanism.

Phosphomevalonate kinase (PMK) catalyzes an essential step in the mevalonate pathway, which is the only pathway for synthesis of isoprenoids and steroids in humans. PMK catalyzes transfer of the gamma-phosphate of ATP to mevalonate 5-phosphate (M5P) to form mevalonate 5-diphosphate. Bringing these phosphate groups in proximity to react is especially challenging, given the high negative charge density on the four phosphate groups in the active site.

Biochemical and structural basis for feedback inhibition of mevalonate kinase and isoprenoid metabolism.

Mevalonate kinase (MK), which catalyzes a key reaction in polyisoprenoid and sterol metabolism in many organisms, is subject to feedback regulation by farnesyl diphosphate and related compounds. The structures of human mevalonate kinase and a binary complex of the rat enzyme incubated with farnesyl thiodiphosphate (FSPP) are reported. Significant FSPP hydrolysis occurs under crystallization conditions; this results in detection of farnesyl thiophosphate (FSP) in the structure of the binary complex.

Functional evaluation of conserved basic residues in human phosphomevalonate kinase.

Phosphomevalonate kinase (PMK) catalyzes the cation-dependent reaction of mevalonate 5-phosphate with ATP to form mevalonate 5-diphosphate and ADP, a key step in the mevalonate pathway for isoprenoid/sterol biosynthesis. Animal PMK proteins belong to the nucleoside monophosphate (NMP) kinase family. For many NMP kinases, multiple basic residues contribute to the neutralization of the negatively charged pentacoordinate phosphate reaction intermediate.

Functional contribution of a conserved, mobile loop histidine of phosphoribulokinase.

In the Rhodobacter sphaeroides phosphoribulokinase (PRK) structure, there are several disordered regions, including a loop containing invariant residues Y98 and H100. The functional importance of these residues has been unclear. PRK is inactivated by diethyl pyrocarbonate (DEPC) and protected by the substrates ATP and Ru5P, as well as by the competitive inhibitor, 6-phosphogluconate, suggesting active site histidine residue(s).

Phosphomevalonate kinase: functional investigation of the recombinant human enzyme.

Phosphomevalonate kinase (PMK) catalyzes a key step in isoprenoid/sterol biosynthesis, converting mevalonate 5-phosphate and ATP to mevalonate 5-diphosphate and ADP. To expedite functional and structural study of this enzyme, an expression plasmid encoding His-tagged human PMK has been constructed and recombinant enzyme isolated in an active, stable form. PMK catalyzes a reversible reaction; kinetic constants of human PMK have been determined for both forward (formation of mevalonate 5-diphosphate) and reverse (formation of mevalonate 5-phosphate) reactions.

Crystal structure of human 3-hydroxy-3-methylglutaryl-CoA Lyase: insights into catalysis and the molecular basis for hydroxymethylglutaric aciduria.

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is a key enzyme in the ketogenic pathway that supplies metabolic fuel to extrahepatic tissues. Enzyme deficiency may be due to a variety of human mutations and can be fatal. Diminished activity has been explained based on analyses of recombinant human mutant proteins or, more recently, in the context of structural models for the enzyme.