2 research outputs found
Site-Directed Mutagenesis of Catalytic Residues in <i>N</i><sup>5</sup>‑Carboxyaminoimidazole Ribonucleotide Synthetase
<i>N</i><sup>5</sup>-CAIR synthetase, an essential enzyme
in microorganisms, converts 5-aminoimidazole ribonucleotide (AIR)
and bicarbonate to <i>N</i><sup>5</sup>-CAIR with the aid
of ATP. Previous X-ray crystallographic analyses of <i>Aspergillus
clavatus N</i><sup>5</sup>-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><sub>i</sub> 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><sup>5</sup>-CAIR synthetase was also detected with ATP displaying
a <i>K</i><sub>i</sub> of 3.3 mM. Taken together, these
results indicate that <i>N</i><sup>5</sup>-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 IC<sub>50</sub> 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