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1.
Mol Cell ; 59(3): 345-358, 2015 Aug 06.
Article in English | MEDLINE | ID: mdl-26145173

ABSTRACT

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). HMGCL expression is upregulated in BRAF V600E-expressing human primary melanoma and hairy cell leukemia cells. Suppression of HMGCL specifically attenuates proliferation and tumor growth potential of human melanoma cells expressing BRAF V600E. Mechanistically, active BRAF upregulates HMGCL through an octamer transcription factor Oct-1, leading to increased intracellular levels of HMGCL product, acetoacetate, which selectively enhances binding of BRAF V600E but not BRAF wild-type to MEK1 in V600E-positive cancer cells to promote activation of MEK-ERK signaling. These findings reveal a mutation-specific mechanism by which oncogenic BRAF V600E "rewires" metabolic and cell signaling networks and signals through the Oct-1-HMGCL-acetoacetate axis to selectively promote BRAF V600E-dependent tumor development.


Subject(s)
Leukemia, Hairy Cell/metabolism , MAP Kinase Kinase 1/metabolism , Melanoma/metabolism , Octamer Transcription Factor-1/metabolism , Oxo-Acid-Lyases/metabolism , Proto-Oncogene Proteins B-raf/metabolism , Signal Transduction , Acetoacetates/metabolism , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans , Mutation , Proto-Oncogene Proteins B-raf/genetics , Up-Regulation
2.
Am J Physiol Regul Integr Comp Physiol ; 308(10): R872-8, 2015 May 15.
Article in English | MEDLINE | ID: mdl-25786485

ABSTRACT

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. DIO rats ate more than DR rats over 3 days of HE diet intake. On day 3 of HE diet intake, DR rats reduced their caloric intake while DIO rats remained hyperphagic. Local VMH astrocyte ketone bodies production was similar between DR and DIO rats during the first 6 h after dark onset feeding but inhibiting VMH ketone body production in DR rats on day 3 transiently returned their intake of HE diet to the level of DIO rats consuming HE diet. In addition, dissociated VMN neurons from DIO and DR rats were equally sensitive to the largely excitatory effects of ß-hydroxybutyrate. Thus while DR rats respond to increased VMH ketone levels by decreasing their intake after 3 days of HE diet, this is not the case of DIO rats. These data suggest that DIO inherent leptin resistance prevents ketone bodies inhibitory action on food intake.


Subject(s)
Energy Intake/physiology , Ketone Bodies/metabolism , Neurons/metabolism , Obesity/metabolism , Ventromedial Hypothalamic Nucleus/metabolism , 3-Hydroxybutyric Acid/pharmacology , Animals , Calcium/metabolism , Diet, High-Fat , Glucose/pharmacology , Male , Neurons/drug effects , Obesity/etiology , Oleic Acid/pharmacology , Rats , Rats, Sprague-Dawley , Ventromedial Hypothalamic Nucleus/drug effects
3.
Arch Biochem Biophys ; 566: 1-6, 2015 Jan 15.
Article in English | MEDLINE | ID: mdl-25499551

ABSTRACT

Mevalonate diphosphate decarboxylase (MDD; EC 4.1.1.33) catalyzes the irreversible decarboxylation of mevalonate diphosphate in the mevalonate pathway to form isopentenyl diphosphate, which is a precursor in the biosynthesis of many essential polyisoprenoid natural products, including sterols. In low G/C Gram-positive bacteria, which utilize the mevalonate pathway, MDD is required for cell viability and thus is a potential target for development of antibiotic drugs. To identify potential inhibitors of the enzyme, the National Cancer Institute's Mechanistic Diversity Set library of compounds was screened for inhibitors of Staphylococcus epidermidis MDD. From this screen, the compound Eriochrome Black A (EBA), an azo dye, was found to inhibit the enzyme with an IC50 value<5µM. Molecular docking of EBA into a crystal structure of S. epidermidis MDD suggested binding at the active site. EBA, along with the related Eriochrome B and T compounds, was evaluated for its ability to not only inhibit enzymatic activity but to inhibit bacterial growth as well. These compounds exhibited competitive inhibition towards the substrate mevalonate diphosphate, with Ki values ranging from 0.6 to 2.7µM. Non-competitive inhibition was observed versus ATP indicating binding of the inhibitor in the mevalonate diphosphate binding site, consistent with molecular docking predictions. Fluorescence quenching analyses also supported active site binding of EBA. These eriochrome compounds are effective at inhibiting S. epidermidis cell growth on both solid media and in liquid culture (MIC50 from 31 to 350µM) raising the possibility that they could be developed into antibiotic leads targeting pathogenic low-G/C Gram-positive cocci.


Subject(s)
Azo Compounds/chemistry , Bacterial Proteins/antagonists & inhibitors , Carboxy-Lyases/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Staphylococcus epidermidis/chemistry , Adenosine Triphosphate/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Carboxy-Lyases/chemistry , Carboxy-Lyases/genetics , High-Throughput Screening Assays , Kinetics , Mevalonic Acid/analogs & derivatives , Mevalonic Acid/chemistry , Molecular Docking Simulation , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Small Molecule Libraries , Staphylococcus epidermidis/enzymology
4.
J Bacteriol ; 196(5): 1055-63, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24375100

ABSTRACT

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. Characterization of these proteins indicated that one enzyme is an isopentenyl phosphate kinase that forms isopentenyl diphosphate (in a reaction analogous to that of Methanococcus jannaschii MJ0044). The second enzyme exhibits a decarboxylase activity that has never been directly attributed to this protein or any homologous protein. It catalyzes the synthesis of isopentenyl phosphate from mevalonate monophosphate, a reaction that has been proposed but never demonstrated by direct experimental proof, which is provided in this account. This enzyme, phosphomevalonate decarboxylase (PMD), exhibits strong inhibition by 6-fluoromevalonate monophosphate but negligible inhibition by 6-fluoromevalonate diphosphate (a potent inhibitor of the classical mevalonate pathway), reinforcing its selectivity for monophosphorylated ligands. Inhibition by the fluorinated analog also suggests that the PMD utilizes a reaction mechanism similar to that demonstrated for the classical MVA pathway decarboxylase. These observations represent the first experimental demonstration in H. volcanii of both the phosphomevalonate decarboxylase and isopentenyl phosphate kinase reactions that are required for an alternate mevalonate pathway in an archaeon. These results also represent, to our knowledge, the first identification and characterization of any phosphomevalonate decarboxylase.


Subject(s)
Carboxy-Lyases/metabolism , Gene Expression Regulation, Enzymologic/physiology , Haloferax volcanii/enzymology , Mevalonic Acid/metabolism , Protein Kinases/metabolism , Amino Acid Sequence , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Carboxy-Lyases/genetics , Catalysis , Gene Expression Regulation, Archaeal/physiology , Haloferax volcanii/genetics , Haloferax volcanii/metabolism , Mevalonic Acid/chemistry , Molecular Sequence Data , Molecular Structure , Protein Kinases/genetics
5.
J Bacteriol ; 195(17): 3854-62, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23794621

ABSTRACT

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 2.3.310). This accomplishment has permitted studies that represent, to the best of our knowledge, the first characterization of an archaeal hydroxymethylglutaryl CoA synthase. Kinetic characterization indicates that, under optimal assay conditions, which include 4 M KCl, the enzyme exhibits catalytic efficiency and substrate saturation at metabolite levels comparable to those reported for the enzyme from nonhalophilic organisms. This enzyme is unique in that it is the first hydroxymethylglutaryl CoA synthase that is insensitive to feedback substrate inhibition by acetoacetyl-CoA. The enzyme supports reaction catalysis in the presence of various organic solvents. Haloferax 3-hydroxy-3-methylglutaryl CoA synthase is sensitive to inactivation by hymeglusin, a specific inhibitor known to affect prokaryotic and eukaryotic forms of the enzyme, with experimentally determined Ki and kinact values of 570 ± 120 nM and 17 ± 3 min(-1), respectively. In in vivo experiments, hymeglusin blocks the propagation of H. volcanii cells, indicating the critical role that the mevalonate pathway plays in isoprenoid biosynthesis by these archaea.


Subject(s)
Cell Membrane/enzymology , Cell Membrane/metabolism , Haloferax volcanii/enzymology , Haloferax volcanii/metabolism , Hydroxymethylglutaryl-CoA Synthase/genetics , Terpenes/metabolism , Acyl Coenzyme A/metabolism , Amino Acid Sequence , Enzyme Inhibitors/metabolism , Gene Expression , Gene Expression Regulation, Archaeal , Gene Expression Regulation, Enzymologic , Haloferax volcanii/genetics , Kinetics , Molecular Sequence Data , Plasmids , Sequence Alignment
6.
J Biol Chem ; 287(40): 33227-36, 2012 Sep 28.
Article in English | MEDLINE | ID: mdl-22865860

ABSTRACT

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. coli produced insoluble material. In contrast, active HMGCLL1 could be recovered upon expression in P. pastoris. Antibodies were prepared against a unique peptide sequence found in the N terminus of the protein. In immunodetection experiments, the antibodies discriminated between HMGCLL1 and mitochondrial HMGCL. Purified enzyme was characterized and demonstrated to cleave HMG-CoA to acetoacetate and acetyl-CoA with catalytic and affinity properties comparable with human mitochondrial HMGCL. The deduced HMGCLL1 sequence contains an N-terminal myristoylation motif; the putative modification site was eliminated by construction of a G2A HMGCLL1. Modification of both proteins was attempted using human N-myristoyltransferase and [(3)H]myristoyl-CoA. Wild-type protein was clearly modified, whereas G2A protein was not labeled. Myristoylation of HMGCLL1 affects its cellular localization. Upon transfection of appropriate expression plasmids into COS1 cells, immunofluorescence detection indicates that G2A HMGCLL1 exhibits a diffuse pattern, suggesting a cytosolic location. In contrast, wild-type HMGCLL1 exhibits a punctate as well as a perinuclear immunostaining pattern, indicating myristoylation dependent association with nonmitochondrial membrane compartments. In control experiments with the HMGCL expression plasmid, protein is localized in the mitochondria, as anticipated. The available results for COS1 cell expression, as well as endogenous expression in U87 cells, indicate that HMGCLL1 is an extramitochondrial hydroxymethylglutaryl-CoA lyase.


Subject(s)
Oxo-Acid-Lyases/chemistry , Acyl Coenzyme A/chemistry , Animals , COS Cells , Catalysis , Cell Line, Tumor , Chlorocebus aethiops , Energy Metabolism , Escherichia coli/metabolism , Female , Humans , Ketone Bodies/chemistry , Ketones , Lipids/chemistry , Lipogenesis , Male , Mitochondria/metabolism , Models, Chemical , Mutagenesis , Neoplasms/metabolism , Oxo-Acid-Lyases/genetics , Peptides/chemistry , Plasmids/metabolism , Rats
7.
Biochem Biophys Res Commun ; 430(1): 313-9, 2013 Jan 04.
Article in English | MEDLINE | ID: mdl-23146631

ABSTRACT

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. Chemical shift changes were monitored, plotted, and fitted to obtain dissociation constants (K(d)). Tight binding compounds with K(d)'s ranging from 6-60 µM were identified. These compounds tended to have significant polarity and negative charge, similar to the natural substrates (M5P and ATP). HSQC cross peak changes suggest that binding induces a global conformational change, such as domain closure. Compounds identified in this study serve as chemical genetic probes of human PMK, to explore pharmacology of the mevalonate pathway, as well as starting points for further drug development.


Subject(s)
Drug Discovery/methods , Enzyme Inhibitors/chemistry , Phosphotransferases (Phosphate Group Acceptor)/antagonists & inhibitors , Catalytic Domain/drug effects , Crystallography, X-Ray , Enzyme Inhibitors/isolation & purification , Enzyme Inhibitors/pharmacology , Humans , Nuclear Magnetic Resonance, Biomolecular , Phosphotransferases (Phosphate Group Acceptor)/chemistry , Protein Structure, Secondary
8.
Biochemistry ; 51(28): 5611-21, 2012 Jul 17.
Article in English | MEDLINE | ID: mdl-22734632

ABSTRACT

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.


Subject(s)
Bacterial Proteins/chemistry , Carboxy-Lyases/chemistry , Mevalonic Acid/analogs & derivatives , Staphylococcus epidermidis/enzymology , Amino Acid Sequence , Binding Sites , Carboxy-Lyases/antagonists & inhibitors , Carboxy-Lyases/genetics , Catalysis , Crystallography, X-Ray , Mevalonic Acid/chemistry , Models, Molecular , Molecular Sequence Data , Mutation , Nucleotides/chemistry , Protein Conformation
9.
Biochemistry ; 51(23): 4713-22, 2012 Jun 12.
Article in English | MEDLINE | ID: mdl-22510038

ABSTRACT

Hymeglusin (1233A, F244, L-659-699) is established as a specific ß-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. Hymeglusin blocks growth of Enterococcus faecalis. After removal of the inhibitor from culture media, a growth curve inflection point at 3.1 h is observed (vs 0.7 h for the uninhibited control). Upon hymeglusin inactivation of purified E. faecalis mvaS, the thioester adduct is more stable than that measured for human HMGCS. Hydroxylamine cleaves the thioester adduct; substantial enzyme activity is restored at a rate that is 8-fold faster for human HMGCS than for mvaS. Structural results explain these differences in enzyme-inhibitor thioester adduct stability and solvent accessibility. The E. faecalis mvaS-hymeglusin cocrystal structure (1.95 Å) reveals virtually complete occlusion of the bound inhibitor in a narrow tunnel that is largely sequestered from bulk solvent. In contrast, eukaryotic (Brassica juncea) HMGCS binds hymeglusin in a more solvent-exposed cavity.


Subject(s)
Enterococcus faecalis/enzymology , Enzyme Inhibitors/pharmacology , Fatty Acids, Unsaturated/pharmacology , Hydroxymethylglutaryl-CoA Synthase/antagonists & inhibitors , Lactones/pharmacology , Cloning, Molecular , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Fatty Acids, Unsaturated/chemistry , Gene Expression Regulation, Bacterial/physiology , Gene Expression Regulation, Enzymologic , Humans , Hydroxylamine/chemistry , Hydroxylamine/pharmacology , Hydroxymethylglutaryl-CoA Synthase/genetics , Hydroxymethylglutaryl-CoA Synthase/metabolism , Kinetics , Lactones/chemistry , Models, Molecular , Molecular Structure , Protein Binding , X-Ray Diffraction
10.
J Biol Chem ; 286(27): 23900-10, 2011 Jul 08.
Article in English | MEDLINE | ID: mdl-21561869

ABSTRACT

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. To facilitate exploration of this possibility, we report the crystal structure of Staphylococcus epidermidis MDD (1.85 Šresolution) and, to the best of our knowledge, the first structures of liganded MDD. These structures include MDD bound to the mevalonate 5-diphosphate analogs diphosphoglycolyl proline (2.05 Šresolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 Šresolution). Comparison of these structures provides a physical basis for the significant differences in K(i) values observed for these inhibitors. Inspection of enzyme/inhibitor structures identified the side chain of invariant Ser(192) as making potential contributions to catalysis. Significantly, Ser → Ala substitution of this side chain decreases k(cat) by ∼10(3)-fold, even though binding interactions between FMVAPP and this mutant are similar to those observed with wild type MDD, as judged by the 2.1 Šcocrystal structure of S192A with FMVAPP. Comparison of microbial MDD structures with those of mammalian counterparts reveals potential targets at the active site periphery that may be exploited to selectively target the microbial enzymes. These studies provide a structural basis for previous observations regarding the MDD mechanism and inform future work toward rational inhibitor design.


Subject(s)
Bacterial Proteins/chemistry , Staphylococcus epidermidis/enzymology , Amino Acid Substitution , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Carboxy-Lyases , Crystallography, X-Ray , Hemiterpenes/chemistry , Hemiterpenes/genetics , Hemiterpenes/metabolism , Mevalonic Acid/analogs & derivatives , Mevalonic Acid/chemistry , Mevalonic Acid/metabolism , Mutation, Missense , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/metabolism , Staphylococcus epidermidis/genetics , Structure-Activity Relationship , Substrate Specificity
11.
J Infect Dis ; 204(4): 609-16, 2011 Aug 15.
Article in English | MEDLINE | ID: mdl-21791663

ABSTRACT

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. METHODS: To identify host or viral protein targets in an unbiased fashion, we performed affinity chromatography, using tandem liquid chromatography/mass spectrometry to identify specific potential targets. RESULTS. Ceestatin binds to 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase and irreversibly inhibits HMG-CoA synthase in a dose-dependent manner. Ceestatin's anti-HCV effects are reversed by addition of HMG-CoA, mevalonic acid, or geranylgeraniol. Treatment with small interfering RNA against HMG-CoA synthase led to a substantial reduction in HCV replication, further validating HMG-CoA synthase as an enzyme essential for HCV replication. CONCLUSIONS: Ceestatin therefore exerts its anti-HCV effects through inhibition of HMG-CoA synthase. It may prove useful as an antiviral agent, as a probe to study HCV replication, and as a cholesterol-lowering agent. The logical stepwise process employed to discover the mechanism of action of ceestatin can serve as a general experimental strategy to uncover the targets on which novel uncharacterized anti-HCV compounds act.


Subject(s)
Antiviral Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Hepacivirus/drug effects , Hydroxymethylglutaryl-CoA Synthase/antagonists & inhibitors , Lactones/pharmacology , Virus Replication/drug effects , Cell Line , Chromatography, Affinity , Hepacivirus/physiology , Humans , Mass Spectrometry , Protein Binding , RNA Interference , RNA, Small Interfering
12.
J Biol Chem ; 285(34): 26341-9, 2010 Aug 20.
Article in English | MEDLINE | ID: mdl-20558737

ABSTRACT

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 2.4 and 2.2 A, respectively. Comparison of these beta/alpha-barrel structures with those of unliganded HMGCL and R41M reveals substantial differences for Mg(2+) coordination and positioning of the flexible loop containing the conserved HMGCL "signature" sequence. In the R41M-Mg(2+)-substrate ternary complex, loop residue Cys(266) (implicated in active-site function by mechanistic and mutagenesis observations) is more closely juxtaposed to the catalytic site than in the case of unliganded enzyme or the WT enzyme-Mg(2+)-3-hydroxyglutaryl-CoA inhibitor complex. In both ternary complexes, the S-stereoisomer of substrate or inhibitor is specifically bound, in accord with the observed Mg(2+) liganding of both C3 hydroxyl and C5 carboxyl oxygens. In addition to His(233) and His(235) imidazoles, other Mg(2+) ligands are the Asp(42) carboxyl oxygen and an ordered water molecule. This water, positioned between Asp(42) and the C3 hydroxyl of bound substrate/inhibitor, may function as a proton shuttle. The observed interaction of Arg(41) with the acyl-CoA C1 carbonyl oxygen explains the effects of Arg(41) mutation on reaction product enolization and explains why human Arg(41) mutations cause drastic enzyme deficiency.


Subject(s)
Acyl Coenzyme A/chemistry , Oxo-Acid-Lyases/chemistry , Arginine , Binding Sites , Crystallography, X-Ray , Humans , Magnesium , Protein Conformation , Water
13.
Arch Biochem Biophys ; 505(2): 131-43, 2011 Jan 15.
Article in English | MEDLINE | ID: mdl-20932952

ABSTRACT

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. The biosynthetic acetoacetyl-CoA thiolase and HMG-CoA synthase reactions rely on key amino acids that are different but are situated in active sites that are similar throughout the family of initial condensation enzymes. Both bacterial and animal HMG-CoA reductases have been extensively studied and the contrasts between these proteins and their interactions with statin inhibitors defined. The conversion of mevalonic acid to isopentenyl 5-diphosphate involves three ATP-dependent phosphorylation reactions. While bacterial enzymes responsible for these three reactions share a common protein fold, animal enzymes differ in this respect as the recently reported structure of human phosphomevalonate kinase demonstrates. There are significant contrasts between observations on metabolite inhibition of mevalonate phosphorylation in bacteria and animals. The structural basis for these contrasts has also recently been reported. Alternatives to the phosphomevalonate kinase and mevalonate diphosphate decarboxylase reactions may exist in archaea. Thus, new details regarding isopentenyl diphosphate synthesis from acetyl-CoA continue to emerge.


Subject(s)
Enzymes/metabolism , Mevalonic Acid/metabolism , Terpenes/metabolism , Animals , Enzyme Inhibitors/pharmacology , Enzymes/chemistry , Humans
14.
Arch Biochem Biophys ; 511(1-2): 48-55, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21514269

ABSTRACT

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. Enzyme activity varies for each cysteine→serine mutant protein and different mutations have widely different effects on recovery of activity upon DTT treatment of non-reduced enzyme. These levels of enzyme activity do not strongly correlate with formation of inter-subunit adducts by these HMGCL mutants. C170S, C266S, and C323S proteins do not form inter-subunit disulfide adducts but such an adduct is restored in the C170S/C174S double mutant. Coexpression of HMGCL proteins encoded by C266S and C323S expression plasmids supports formation of a C266S/C323S heterodimer which does form a covalent inter-subunit adduct. These observations are interpreted in the context of competition between cysteines in formation of intra-subunit and inter-subunit heterodisulfide adducts.


Subject(s)
Oxo-Acid-Lyases/chemistry , Oxo-Acid-Lyases/metabolism , Amino Acid Sequence , Amino Acid Substitution , Base Sequence , Cysteine/chemistry , DNA Primers/genetics , Dimerization , Humans , In Vitro Techniques , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Oxo-Acid-Lyases/genetics , Protein Multimerization , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid
15.
J Am Chem Soc ; 132(7): 2102-3, 2010 Feb 24.
Article in English | MEDLINE | ID: mdl-20112895

ABSTRACT

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. However, the way in which this charge neutralization and binding is achieved, from a structural and dynamics perspective, is not known. More broadly, the role of arginine side-chain dynamics in binding of charged substrates has not been experimentally defined for any protein to date. Herein we report a characterization of changes to the dynamical state of the arginine side chains in PMK due to binding of its highly charged substrates, ATP and M5P. These studies were facilitated by the use of arginine-selective labeling to eliminate spectral overlap. Model-free analysis indicated that while substrate binding has little effect on the arginine backbone dynamics, binding of either substrate leads to significant rigidification of the arginine side chains throughout the protein, even those that are >8 A from the binding site. Such a global rigidification of arginine side chains is unprecedented and suggests that there are long-range electrostatic interactions of sufficient strength to restrict the motion of arginine side chains on the picosecond-to-nanosecond time scale. It will be interesting to see whether such effects are general for arginine residues in proteins that bind highly charged substrates, once additional studies of arginine side-chain dynamics are reported.


Subject(s)
Arginine/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Phosphotransferases (Phosphate Group Acceptor)/chemistry , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Arginine/metabolism , Humans , Ligands , Mevalonic Acid/analogs & derivatives , Mevalonic Acid/chemistry , Mevalonic Acid/metabolism , Models, Molecular , Phosphotransferases (Phosphate Group Acceptor)/metabolism , Thermodynamics
16.
Anal Biochem ; 396(1): 96-102, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19706283

ABSTRACT

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. The assay uses dithiobisnitrobenzoic acid (DTNB) to detect coenzyme A (CoASH) release on acetylation of enzyme by the substrate acetyl-CoA, which precedes condensation with acetoacetyl-CoA to form the HMG-CoA product. The assay method takes advantage of the stability of recombinant enzyme in the absence of a reducing agent. It can be scaled down to a 60 microl volume to allow the use of 384-well microplates, facilitating high-throughput screening of compound libraries. Enzyme activity measured in the microplate assay is comparable to values measured by using conventional scale spectrophotometric assays with the DTNB method (412 nm) for CoASH production or by monitoring the use of a second substrate, acetoacetyl-CoA (300 nm). The high-throughput assay method has been successfully used to screen a library of more than 100,000 drug-like compounds and has identified both reversible and irreversible inhibitors of the human enzyme.


Subject(s)
Enzyme Assays/methods , High-Throughput Screening Assays/methods , Hydroxymethylglutaryl-CoA Synthase/analysis , Light , Spectrophotometry/methods , Acetyl Coenzyme A/metabolism , Dithionitrobenzoic Acid/metabolism , Enzyme Activation/drug effects , Enzyme Inhibitors/analysis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Hydroxymethylglutaryl-CoA Synthase/antagonists & inhibitors , Hydroxymethylglutaryl-CoA Synthase/metabolism , Kinetics , Reproducibility of Results , Small Molecule Libraries/analysis , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Substrate Specificity/drug effects , Terpenes/metabolism , Time Factors
17.
Proteins ; 75(1): 127-38, 2009 Apr.
Article in English | MEDLINE | ID: mdl-18798562

ABSTRACT

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. As such, conformational and dynamics changes needed to form the Michaelis complex are of mechanistic interest. Herein, we report the characterization of substrate induced changes (Mg-ADP, M5P, and the ternary complex) in PMK using NMR-based dynamics and chemical shift perturbation measurements. Mg-ADP and M5P K(d)'s were 6-60 microM in all complexes, consistent with there being little binding synergy. Binding of M5P causes the PMK structure to compress (tau(c) = 13.5 nsec), whereas subsequent binding of Mg-ADP opens the structure up (tau(c) = 15.6 nsec). The overall complex seems to stay very rigid on the psec-nsec timescale with an average NMR order parameter of S(2) approximately 0.88. Data are consistent with addition of M5P causing movement around a hinge region to permit domain closure, which would bring the M5P domain close to ATP to permit catalysis. Dynamics data identify potential hinge residues as H55 and R93, based on their low order parameters and their location in extended regions that connect the M5P and ATP domains in the PMK homology model. Likewise, D163 may be a hinge residue for the lid region that is homologous to the adenylate kinase lid, covering the "Walker-A" catalytic loop. Binding of ATP or ADP appears to cause similar conformational changes; however, these observations do not indicate an obvious role for gamma-phosphate binding interactions. Indeed, the role of gamma-phosphate interactions may be more subtle than suggested by ATP/ADP comparisons, because the conservative O to NH substitution in the beta-gamma bridge of ATP causes a dramatic decrease in affinity and induces few chemical shift perturbations. In terms of positioning of catalytic residues, binding of M5P induces a rigidification of Gly21 (adjacent to the catalytically important Lys22), although exchange broadening in the ternary complex suggests some motion on a slower timescale does still occur. Finally, the first nine residues of the N-terminus are highly disordered, suggesting that they may be part of a cleavable signal or regulatory peptide sequence.


Subject(s)
Mevalonic Acid/analogs & derivatives , Phosphotransferases (Phosphate Group Acceptor)/chemistry , Phosphotransferases (Phosphate Group Acceptor)/metabolism , Adenosine Triphosphate/metabolism , Humans , Mevalonic Acid/metabolism , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Conformation , Substrate Specificity
18.
Arch Biochem Biophys ; 480(1): 58-67, 2008 Dec 01.
Article in English | MEDLINE | ID: mdl-18823933

ABSTRACT

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)=6.1+/-0.5 U/mg; K(m) for ATP is 0.69+/-0.07 mM and K(m) for (R,S) mevalonate diphosphate is 28.9+/-3.3 microM. Conserved polar residues predicted to be in the hMDD active site were mutated to test functional importance. R161Q exhibits a approximately 1000-fold diminution in specific activity, while binding the fluorescent substrate analog, TNP-ATP, comparably to wild-type enzyme. Diphosphoglycolyl proline (K(i)=2.3+/-0.3 uM) and 6-fluoromevalonate 5-diphosphate (K(i)=62+/-5 nM) are competitive inhibitors with respect to mevalonate diphosphate. N17A exhibits a V(max)=0.25+/-0.0 2U/mg and a 15-fold inflation in K(m) for mevalonate diphosphate. N17A's K(i) values for diphosphoglycolyl proline and fluoromevalonate diphosphate are inflated (>70-fold and 40-fold, respectively) in comparison with wild-type enzyme. hMDD structure indicates the proximity (2.8A) between R161 and N17, which are located in an interior pocket of the active site cleft. The data suggest the functional importance of R161 and N17 in the binding and orientation of mevalonate diphosphate.


Subject(s)
Carboxy-Lyases/chemistry , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Base Sequence , Binding, Competitive , Carboxy-Lyases/genetics , Carboxy-Lyases/metabolism , Catalytic Domain/genetics , Crystallography, X-Ray , DNA Primers/genetics , Humans , In Vitro Techniques , Kinetics , Mevalonic Acid/analogs & derivatives , Mevalonic Acid/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid
19.
Protein Sci ; 15(4): 837-42, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16522805

ABSTRACT

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). PRK contains only three invariant histidines: H45, H100, and H134. Previous mutagenesis studies discount significant function for H134, but implicate H45 in Ru5P binding. PRK mutant H45N is inactivated by DEPC, implicating a second active site histidine. To evaluate the function of H100, as well as another invariant loop residue Y98, PRK mutants Y98L, H100A, H100N, and H100Q were characterized. Mutant PRK binding stoichiometries for the fluorescent alternative substrate, trinitrophenyl-ATP, as well as the allosteric activator, NADH, are comparable to wild-type PRK values, suggesting intact effector and substrate binding sites. The K(mRu5P) for the H100 mutants shows modest eight- to 14-fold inflation effects, whereas Y98L exhibits a 40-fold inflation for K(mRu5P). However, Y98L's K(i) for the competitive inhibitor 6-phosphogluconate is close to that of wild-type PRK. These observations suggest that Y98 and H100 are not essential Ru5P binding determinants. The Vm of Y98L is diminished 27-fold compared with wild-type PRK. In contrast, H100A, H100N, and H100Q exhibit significant decreases in Vm of 2600-, 2300-, and 735-fold, respectively. Results suggest that the mobile region containing Y98 and H100 must contribute to PRK's active site. Moreover, H100's imidazole significantly influences catalytic efficiency.


Subject(s)
Histidine/chemistry , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Amino Acid Sequence , Binding Sites , Conserved Sequence , Diethyl Pyrocarbonate/chemistry , Diethyl Pyrocarbonate/metabolism , Evaluation Studies as Topic , Histidine/metabolism , Kinetics , Molecular Sequence Data , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/isolation & purification , Point Mutation , Protein Structure, Secondary , Sequence Alignment
20.
J Mol Model ; 22(1): 13, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26680991

ABSTRACT

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. The validated docking protocol was then used to predict structures of the inhibitors bound to Staphylococcus aureus MDD using the unliganded crystal structure of Staphylococcus aureus MDD. We also investigated a possible interactions improvement by combining this docking method with molecular dynamics simulations. Thus, the predicted docking structures were analyzed in a molecular dynamics trajectory to generate dynamic models and reinforce the predicted binding modes. FMVAPP is predicted to make more extensive contacts with S. aureus MDD, forming stable hydrogen bonds with Arg144, Arg193, Lys21, Ser107, and Tyr18, as well as making stable hydrophobic interactions with Tyr18, Trp19, and Met196. The differences in predicted binding are supported by experimentally determined Ki values of 0.23 ± 0.02 and 34 ± 8 µM, for FMVAPP and DPGP, respectively. The structural information coupled with the kinetic characterization obtained from this study should be useful in defining the requirements for inhibition as well as in guiding the selection of active compounds for inhibitor optimization.


Subject(s)
Bacterial Proteins/chemistry , Carboxy-Lyases/chemistry , Molecular Docking Simulation , Molecular Dynamics Simulation , Staphylococcus aureus/enzymology , Catalytic Domain
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