Abstract
Antibiotic resistance is becoming an increasing problem in the treatment of bacterial pathogens. A deeper understanding of inosine 5′-monophosphate dehydrogenase (IMPDH) inhibition at a biochemical level may reveal a route to the development of novel antimicrobial drugs. IMPDH catalyzes the oxidation of IMP to XMP, the first committed and rate-limiting step in de novo guanine nucleotide biosynthesis. Several inhibitors of bacterial IMPDH have been developed. Although all bacterial IMPDHs sensitive to these inhibitors have highly conserved inhibitor-binding sites, there is still quite a bit of variation in inhibitor sensitivity between the IMPDHs of various bacterial species. Our goal is to determine which residues within the IMPDH protein affect sensitivity to inhibitors. Specifically, we hypothesized that mutations in the dynamic flap region of Bacillus anthracis IMPDH (BaIMPDH) would alter its sensitivity to inhibitors. Several point mutations were incorporated into BaIMPDH through site-directed mutagenesis (SDM) and mutant proteins were expressed and purified from E. coli with nickel affinity chromatography. The mutants were analyzed with both kinetic and IC50 studies, and compared to wild type BaIMPDH. It was found that the kinetic parameters of the mutants were similar to those of wild type, indicating that the mutants have not greatly disrupted enzymatic activity. IC50 fold changes were constant for some inhibitors and different for others, suggesting that the flap residues both influence conformational equilibrium between the open-flap and closed-flap conformations, as well as interact with some inhibitors. Further studies will correlate inhibitor structures with IC50 fold changes.