| In an effort to provide mechanistic and structural information on VanX, the Zn(II)-containing dipeptidase required for high-level vancomycin resistance, a combination of kinetic, mechanistic, and spectroscopic studies were conducted. Three strategies were used to overexpress and purify recombinant VanX from E. coli cells: (1) a pET-5b-based system, (2) an IMPACT-T7-based system, and (3) a pET-27b-based system. While all three systems resulted in large amounts (>10 mg/L of culture) of VanX, the pET-27b-based system was judged to be superior.;The development of a novel, continuous UV-Vis spectrophotometric assay using L-alanine-p-nitroanilide as the substrate is presented. Results from steady-state kinetic and inhibition studies were compared to previously published data obtained using a different assay to confirm the reliability of the continuous assay. When using this assay, pH dependence and solvent isotope studies revealed a kinetically significant proton transfer during the hydrolysis of substrate. A minimal kinetic mechanism accounting for the results obtained using the continuous assay.;Structural characterization using MALDI-TOF mass spectrometry revealed that VanX exists primarily as a dimer in solution; however, monomeric and tetrameric forms are also present. Circular dichroism studies revealed that the secondary structure of the overexpressed recombinant VanX samples were of the alpha + beta classification, consistent with the X-ray crystal structure. EPR and NMR spectroscopic investigations of Co(II)-, and Cu(II)-substituted VanX revealed that these techniques could be used to probe binding and aid in the characterization of potentially, clinically useful inhibitors. |
