Site-Directed Mutagenesis of Catalytic Residues in <i>N</i>⁵‑Carboxyaminoimidazole Ribonucleotide Synthetase

<i>N</i>⁵-CAIR synthetase, an essential enzyme in microorganisms, converts 5-aminoimidazole ribonucleotide (AIR) and bicarbonate to <i>N</i>⁵-CAIR with the aid of ATP. Previous X-ray crystallographic analyses of <i>Aspergillus clavatus N</i>⁵-CAIR synthetase postulated that R271, H273, and K353 were important for bicarbonate binding and for catalysis. As reported here, site-directed mutagenesis of these residues revealed that R271 and H273 are, indeed, critical for bicarbonate binding and catalysis whereas all K353 mutations, even ones conservative in nature, are inactive. Studies on the R271K mutant protein revealed cooperative substrate inhibition for ATP with a <i>K</i>_i of 1.2 mM. Kinetic investigation of the H273A mutant protein indicated that it was cooperative with respect to AIR; however, this effect was not seen in either the wild-type or any of the other mutant proteins. Cooperative ATP-dependent inhibition of wild-type <i>N</i>⁵-CAIR synthetase was also detected with ATP displaying a <i>K</i>_i of 3.3 mM. Taken together, these results indicate that <i>N</i>⁵-CAIR synthetase operates maximally within a narrow concentration of ATP

The Design, Synthesis, and Characterizations of Spore Germination Inhibitors Effective against an Epidemic Strain of Clostridium difficile

Clostridium difficile infections (CDI), particularly those caused by the BI/NAP1/027 epidemic strains, are challenging to treat. One method to address this disease is to prevent the development of CDI by inhibiting the germination of C. difficile spores. Previous studies have identified cholic amide <i>m</i>-sulfonic acid, CamSA, as an inhibitor of spore germination. However, CamSA is inactive against the hypervirulent strain R20291. To circumvent this problem, a series of cholic acid amides were synthesized and tested against R20291. The best compound in the series was the simple phenyl amide analogue which possessed an IC₅₀ value of 1.8 μM, more than 225 times as potent as the natural germination inhibitor, chenodeoxycholate. This is the most potent inhibitor of C. difficile spore germination described to date. QSAR and molecular modeling analysis demonstrated that increases in hydrophobicity and decreases in partial charge or polar surface area were correlated with increases in potency