The Kinetic Mechanism of Phosphomevalonate Kinase

Pilloff, Daniel E.; Dabovic, Kristina; Romanowski, M.J.; Bonanno, J.B.; Doherty, Mary K.; Burley, S.K.; Leyh, Thomas S.

doi:10.1074/jbc.m210551200

Cited by 52 publications

(60 citation statements)

References 35 publications

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“…liver mevalonate kinase (42) and liver MDD (43). Yet others still appear to utilize a random order of substrate binding in proceeding to a ternary reaction complex (E. coli galactokinase (44) and Streptococcus pneumoniae phosphomevalonate kinase (45). Based upon the crystal structures presented here, it seems unlikely that MDD could function through any mechanism other than binding the MVAPP acceptor prior to ATP.…”

Section: Discussionmentioning

confidence: 99%

Crystal Structures of Staphylococcus epidermidis Mevalonate Diphosphate Decarboxylase Bound to Inhibitory Analogs Reveal New Insight into Substrate Binding and Catalysis

Barta

Skaff

McWhorter

et al. 2011

Journal of Biological Chemistry

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The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in Grampositive 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 Å resolution) 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 Å resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 Å resolution). 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 3 Ala substitution of this side chain decreases k cat by ϳ10 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 Å cocrystal 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.The ever-growing trend among many bacterial pathogens toward antibiotic resistance represents one of the single greatest threats to public health in both developing and modern nations. In particular, a growing body of literature from the last decade has demonstrated that many strains of the widespread Gram-positive organisms Staphylococcus aureus and Staphylococcus epidermidis are now insensitive toward an array of the ␤-lactam class antibiotics that were once considered frontline therapeutics (1, 2). As recently as a few years ago, the problem of antibiotic resistance was associated primarily with those infections arising from within the healthcare setting. However, recent studies have shown that resistant strains are now spreading rapidly within the community, where they may cause potentially life-threatening illness in persons not recently hospitalized or undergoing invasive medical procedures (1, 3). Given the limited nature of effective therapeutic tools to combat these diseases, all such infections must be carefully managed to prevent further spread throughout the population. As a consequence, there is now renewed interest in novel classes of antimicrobials that are effective against sensitive and...

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Section: Discussionmentioning

confidence: 99%

Crystal Structures of Staphylococcus epidermidis Mevalonate Diphosphate Decarboxylase Bound to Inhibitory Analogs Reveal New Insight into Substrate Binding and Catalysis

Barta

Skaff

McWhorter

et al. 2011

Journal of Biological Chemistry

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“…General Materials and Methods-Lactate dehydrogenase (LDH, rabbit muscle), pyruvate kinase (PK, rabbit muscle), and alkaline phosphatase (calf intestine) were purchased from Roche Applied Science and treated as described previously (11). ATP, ADP, NADH, phosphoenolpyruvate (PEP), ␤-mercaptoethanol (2-ME), (R,S)-mevalonate (Mev), and triethylamine were purchased from Sigma.…”

Section: Methodsmentioning

confidence: 99%

“…Competent Escherichia coli BL21(DE3) was purchased from Novagen. Racemic mevalonolactone analogues ((R,S)-X), Streptococcus pneumoniae MK, PMK, DPM-DC, and Staphylococcus aureus MK were prepared as described previously (9,11). Chromatography was carried out using an Ä KTA fast-protein liquid chromatography system (Amersham Biosciences).…”

Section: Methodsmentioning

confidence: 99%

Probing Ligand-binding Pockets of the Mevalonate Pathway Enzymes from Streptococcus pneumoniae

Lefurgy

Rodriguez

Park

et al. 2010

Journal of Biological Chemistry

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Diphosphomevalonate (Mev⅐pp) is the founding member of a new class of potential antibiotics targeting the Streptococcus pneumoniae mevalonate (Mev) pathway. We have synthesized a series of Mev⅐pp analogues designed to simultaneously block two steps in this pathway, through allosteric inhibition of mevalonate kinase (MK) and, for five of the analogues, by mechanismbased inactivation of diphosphomevalonate decarboxylase (DPM-DC). The analogue series expands the C 3 -methyl group of Mev⅐pp with hydrocarbons of varying size, shape, and chemical and physical properties. Previously, we established the feasibility of a prodrug strategy in which unphosphorylated Mev analogues could be enzymatically converted to the active Mev⅐pp forms by the endogenous MK and phosphomevalonate kinase. We now report the kinetic parameters for the turnover of non-, mono-, and diphosphorylated analogues as substrates and inhibitors of the three mevalonate pathway enzymes. The inhibition of MK by Mev⅐pp analogues revealed that the allosteric site is selective for compact, electron-rich C 3 -subsitutents. The lack of reactivity of analogues with DPM-DC provided evidence, counter to the existing model, for a decarboxylation transition state that is concerted rather than dissociative. The Mev pathway is composed of three structurally and functionally conserved enzymes that catalyze consecutive steps in a metabolic pathway. The current work reveals that these enzymes exhibit significant differences in specificity toward R-group substitution at C 3 and that these patterns are explained well by changes in the volume of the C 3 R-group-binding pockets of the enzymes.Streptococcus pneumoniae, the primary cause of bacterial meningitis and pneumonia, kills over 4000 people daily worldwide and disproportionately affects children and the elderly (1, 2). Antibiotic resistance is a major problem in fighting this organism, with multiple-drug resistance rates as high as 95% in some regions (3). Although vaccines targeting the 7 or 23 most prevalent strains have shown success in reducing disease incidence in developed countries (4), there is a continual need for new antibiotic strategies to combat unvaccinated strains, which are rapidly filling the biological niches left by the vaccine (5).The mevalonate (Mev) 3 pathway is essential for the survival of S. pneumoniae in lung and serum (6). The bacterium uses this pathway to convert Mev to isopentenyl diphosphate, the "building block" of the isoprenoids: a class of 25,000 unique molecules having a wide range of biological functions. The pathway consists of three GHMP family kinases: Mev kinase (MK), phosphomevalonate kinase (PMK) and diphosphomevalonate decarboxylase (DPM-DC) ( Fig. 1) (7). Mutations that knock out genes in this pathway kill the organism in vivo, suggesting that S. pneumoniae cannot obtain the necessary precursors or downstream products from the host (6). In principle, each of the three enzymes is an antibiotic target, because inhibition of any of them prevents the production of isopentenyl d...

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“…However, the function of the mevalonate pathway in these bacteria is supported by characterization of Enterococcus faecalis 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase (20), as well as a dual-function protein, acetoacetyl-CoA thiolase/HMG-CoA reductase (9). Recently, phosphomevalonate kinase activity has been observed for a protein from Streptococcus pneumoniae (15). In contrast, there has not been substantial characterization of mevalonate kinase isolated from any grampositive coccus.…”

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confidence: 99%

Staphylococcus aureus Mevalonate Kinase: Isolation and Characterization of an Enzyme of the Isoprenoid Biosynthetic Pathway

2004

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It has been proposed that isoprenoid biosynthesis in several gram-positive cocci depends on the mevalonate pathway for conversion of acetyl coenzyme A to isopentenyl diphosphate. Mevalonate kinase catalyzes a key reaction in this pathway. In this study the enzyme from Staphylococcus aureus was expressed in Escherichia coli, isolated in a highly purified form, and characterized. The overall amino acid sequence of this enzyme was very heterologous compared with the sequences of eukaryotic mevalonate kinases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration chromatography suggested that the native enzyme is a monomer with a molecular mass of approximately 33 kDa. The specific activity was 12 U/mg, and the pH optimum was 7.0 to 8.5. The apparent K m values for R,S-mevalonate and ATP were 41 and 339 M, respectively. There was substantial substrate inhibition at millimolar levels of mevalonate. The sensitivity to feedback inhibition by farnesyl diphosphate and its sulfur-containing analog, farnesyl thiodiphosphate, was characterized. These compounds were competitive inhibitors with respect to ATP; the K i values were 46 and 45 M for farnesyl diphosphate and its thio analog, respectively. Parallel measurements with heterologous eukaryotic mevalonate kinases indicated that S. aureus mevalonate kinase is much less sensitive to feedback inhibition (K i difference, 3 orders of magnitude) than the human enzyme. In contrast, both enzymes tightly bound trinitrophenyl-ATP, a fluorescent substrate analog, suggesting that there are similarities in structural features that are important for catalytic function.During isoprenoid biosynthesis in most eubacteria the methyl erythritol 4-phosphate pathway is used for production of isopentenyl diphosphate. In contrast, animals, yeast, and archaea produce isopentenyl diphosphate by the better-characterized mevalonate pathway. Recently, it has been reported (23) that the genomes of several gram-positive cocci encode enzymes of the mevalonate pathway and that survival of these bacteria requires this pathway. The genes that have been proposed to encode enzymes of the mevalonate pathway in grampositive cocci are heterologous with the coding sequences for comparable enzymes in higher organisms. However, the function of the mevalonate pathway in these bacteria is supported by characterization of Enterococcus faecalis 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase (20), as well as a dual-function protein, acetoacetyl-CoA thiolase/HMG-CoA reductase (9). Recently, phosphomevalonate kinase activity has been observed for a protein from Streptococcus pneumoniae (15). In contrast, there has not been substantial characterization of mevalonate kinase isolated from any grampositive coccus.Mevalonate kinase (EC 2.7.1.36) catalyzes the following reaction (the reaction is divalent cation dependent, and Mn This enzyme is a key enzyme (22) in the mevalonate pathway for biosynthesis of isopentenyl diphosphate. In fact, genetic defects that decrease ...

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The Kinetic Mechanism of Phosphomevalonate Kinase

Cited by 52 publications

References 35 publications

Crystal Structures of Staphylococcus epidermidis Mevalonate Diphosphate Decarboxylase Bound to Inhibitory Analogs Reveal New Insight into Substrate Binding and Catalysis

Crystal Structures of Staphylococcus epidermidis Mevalonate Diphosphate Decarboxylase Bound to Inhibitory Analogs Reveal New Insight into Substrate Binding and Catalysis

Probing Ligand-binding Pockets of the Mevalonate Pathway Enzymes from Streptococcus pneumoniae

Staphylococcus aureus Mevalonate Kinase: Isolation and Characterization of an Enzyme of the Isoprenoid Biosynthetic Pathway

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