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 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 → 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 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.
Highlights
AI071028 and AI090149. □S The on-line version of this article contains supplemental Figs
To clarify previous observations regarding the mevalonate diphosphate decarboxylase (MDD) mechanism and to better understand the basis for substrate recognition by MDD proteins, we report here the high resolution x-ray crystal structures of S. epidermidis MDD both free and bound to the inhibitory substrate analogs diphosphoglycolyl proline (DPGP) and 6-fluoromevalonate diphosphate (FMVAPP)
MDD structures were obtained from the PDB [31] and are as follows: S. cerevisiae (1FI4); S. pyogenes (2GS8); S. aureus (2HK2 and 2HK3); T. brucei (2HKE); H. sapiens (3DJ4); M. musculus (3F0N); L. pneumophila (3LTO)
Summary
Overexpression, and Purification of Recombinant Forms of MDD—A gene fragment encoding the entire open reading frame (residues 1–327) of MDD was amplified from S. epidermidis genomic DNA via PCR and subcloned into the expression plasmid pT7HMT [14]. The purified protein was concentrated to 5 mg/ml and buffer exchanged by ultrafiltration into 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, and stored at 4 °C for further use. The pH was adjusted to ϳ8.0 with cold HCl, and the concentration of the physiologically active R isomer was determined using MDD for an enzymatic end point assay. Optimal data fits (R2 Ͼ0.97) were obtained using either competitive (FMVAPP) or mixed (DPGP) inhibition models. A final cycle of TLS refinement was used to complete the structure of wild type MDD bound to FMVAPP. In all cases reported here, inspection of the initial Fo Ϫ Fc maps described above revealed unmodeled contiguous density that corresponded to ordered ligand in the active site of both copies of MDD found within the asymmetric unit.
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