A Fluorescence‐Based Assay for N5‐Carboxyaminoimidazole Ribonucleotide Mutase

A robust, fluorescence-based assay has been developed that relies on the reaction between the novel istain-fluorecein conjugate and the enzymatically generated compound, AIR. These compounds react non-enzymatically resulting in a highly fluorescent product. This unique reaction provides a new assay for monitoring the activity of the enzyme N⁵-CAIR mutase.

Abstract

The 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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