Functional and structural characterization of the mevalonate diphosphate decarboxylase and the isopentenyl diphosphate isomerase from Enterococcus faecalis
Enterococcus faecalis causes a diverse range of nosocomial infections (in wounds, the gastrointestinal tract, the blood stream and the endocardium), and multidrug-resistant strains have become a serious issue across countries. Vancomycin, a FDA-approved drug for the disruption of the bacterial cell wall biosynthesis, has been utilized to treat infectious diseases caused by Enterococci; however, the prevalence of vancomycin-resistant enterococci (VRE) threatens communities all over the world. We aim at developing novel therapeutic strategies to control bacterial growth of Enterococci, and we focus on targeting two essential enzymes involved in poly-isoprenoid biosynthesis in Enterococcus faecalis; one is the mevalonate diphosphate decarboxylase (MDD) in the mevalonate pathway, and the other one is the isopentenyl diphosphate isomerase (IDI), which is downstream of MDD. Functional and structural studies on the mevalonate diphosphate decarboxylase from Enterococcus faecalis (MDDEF) have been conducted using enzymology, isothermal titration calorimetry (ITC) and X-ray crystallography. We have used classical enzymology approaches to determine the substrate binding mechanism of MDDEF, which belongs to a compulsory bi-substrate mechanism with mevalonate diphosphate (MVAPP) binding first. We also obtained thermodynamic data on substrate binding in MDDEF using ITC. The results have suggested that enhanced binding of the second substrate, ATP, can be achieved by the prerequisite binding of MVAPP. In structure determination, we obtained several crystal structures of MDDEF (with or without ligand binding), which represent different states in the enzymatic reaction. Based on structural comparisons, structural changes upon substrate binding can be observed. Conformational changes in two non-conserved regions during the substrate binding event may suggest unique approaches to structure-based specific drug development in the near future. In the complex structure of MDDEF bound with MVAPP, ADPBeF3 and Mg2+, the ordering of two regions (97-104 and 183-191) produces a closed conformation which represents a transitional pre-phosphoryl transfer state. The ligand architecture implicates a dissociative phosphoryl transfer mechanism during the chemical steps of the enzymatic reaction. A high-throughput screening method for identification of inhibitors against MDDEF has been established, and the human MDD protein has been successfully expressed via auto-induction and purified to homogeneity. Human MDD will serve as a selection marker for elimination of non-specific compounds targeting the conserved active-site region in MDD proteins. We have also successfully expressed and purified the isopentenyl diphosphate isomerase from E. faecalis (IDI-2EF), and have obtained crystallization conditions and optimized them for crystallizing apo-form IDI-2EF. Diffraction data were collected at the APS synchrotron source where the apo-form crystal of IDI-2 EF diffracted to a 2.0-Å resolution. We are doing experiments to obtain the bound form of IDI-2EF. These structures can be further utilized for structure-based drug design targeting IDI-2EF in the future. In summary, our new findings in MDDEF and IDI-2EF will provide the detailed enzyme mechanisms and insight into new therapeutic strategies against multi-drug resistant enterococci.
Stauffacher, Purdue University.
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