Marcella F. Sharma, Shiv K. Sharma, Cale C. Streeter, Steven M. Firestine

A Fluorescence‐Based Assay for N5‐Carboxyaminoimidazole Ribonucleotide Mutase

  • Organic Chemistry
  • Molecular Biology
  • Molecular Medicine
  • Biochemistry

AbstractThe enzyme N5‐carboxylaminoinidazole ribonucleotide (N5‐CAIR) mutase is found in microbial de novo purine biosynthesis but is absent in humans making it an attractive antimicrobial target. N5‐CAIR mutase catalyzes the synthesis of carboxyaminoimidazole ribonucleotide (CAIR) from N5‐CAIR which is itself prepared from aminoimidazole ribonucleotide (AIR) by the enzyme N5‐CAIR synthetase. During our research on identifying inhibitors of N5‐CAIR mutase, we developed an innovative, fluorescence‐based assay to measure the activity of this enzyme. This assay relies upon our recent serendipitous observation that AIR reversibly reacts with the compound isatin. Reaction of a fluorescently‐tagged isatin with AIR resulted in a large increase in fluorescence intensity allowing a measurement of the concentration of AIR in solution. From this observation, we developed a reproducible, non‐continuous assay that can replicate the known kinetic parameters of the enzyme and can readily detect a recognized inhibitor of the enzyme. This assay should find utility in screening for inhibitors targeting N5‐CAIR mutase.

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