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

Abstract

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...

Identification in Haloferax volcanii of Phosphomevalonate Decarboxylase and Isopentenyl Phosphate Kinase as Catalysts of the Terminal Enzyme Reactions in an Archaeal Alternate Mevalonate Pathway

“…This gap omits serine and arginine residues that have been implicated in binding the beta-phosphoryl of mevalonate diphosphate in diphosphatespecific MDDs. In fact, these residues make multiple interactions with this phosphoryl moiety (16). In the context of the absence of these residues in the PMD "gap," it will be interesting to determine whether, in other archaea that encode an IPK enzyme, there are also proteins annotated as MDD enzymes while functioning as PMD enzymes.…”

In Vivo Formation of the Protein Disulfide Bond That Enhances the Thermostability of Diphosphomevalonate Decarboxylase, an Intracellular Enzyme from the Hyperthermophilic Archaeon Sulfolobus solfataricus

“…S. solfataricus is the only example of archaea that has been proven to date to possess the classical MVA pathway (22). The crystal structures of DMD have been solved with the enzymes from several eukaryotes (Saccharomyces cerevisiae [27], Trypanosoma brucei [28], Homo sapiens [29], and Mus musculus), and bacteria (Staphylococcus aureus [28], Staphylococcus epidermidis [30,31], Streptococcus pyogenes, and Legionella pneumophila). Most of the known DMD structures have been reported as homodimers, as elucidated biochemically for some eukaryotic enzymes (32)(33)(34), while DMD from Trypanosoma brucei was shown to be monomeric (28).…”

A structurally dynamic N-terminal region drives function of the staphylococcal peroxidase inhibitor (SPIN)

“…Briefly, recombinant strains of E. coli BL21(DE3) bearing a plasmid of interest were grown in 1 L of selective Terrific Broth at 37 ºC, prior to inducing protein expression with 1 mM IPTG at 18 ºC overnight. Cells from the induced culture were harvested by centrifugation, resuspended, lysed by microfluidization, and processed for Ni-NTA affinity chromatography as previously described (33). Following initial purification, the recombinant SPIN proteins were digested with TEV protease to remove the affinity tag as described elsewhere (32).…”