A Fluorescence-Based Assay for N⁵ -Carboxyaminoimidazole Ribonucleotide Mutase

Area of Science:

• Biochemistry
• Enzymology
• Microbial Metabolism

Background:

• N5-carboxylaminoimidazole ribonucleotide (N5-CAIR) mutase is crucial for microbial purine biosynthesis but absent in humans.
• This enzyme presents a promising target for antimicrobial drug development.
• N5-CAIR mutase catalyzes the conversion of N5-CAIR to carboxyaminoimidazole ribonucleotide (CAIR).

Purpose of the Study:

• To develop an innovative, fluorescence-based assay for measuring N5-CAIR mutase activity.
• To validate the assay's ability to replicate known enzyme kinetics and detect inhibitors.
• To facilitate the screening of potential N5-CAIR mutase inhibitors.

Main Methods:

• A novel fluorescence assay was developed based on the reaction between aminoimidazole ribonucleotide (AIR) and a fluorescently tagged isatin derivative.
• The assay measures the increase in fluorescence intensity upon AIR binding to isatin.
• The assay's performance was validated by replicating known kinetic parameters and detecting a known inhibitor.

Main Results:

• The developed assay demonstrated reproducibility and accurately reflected the enzyme's known kinetic parameters.
• The assay successfully detected a known inhibitor of N5-CAIR mutase.
• The assay provides a sensitive method for quantifying AIR concentration.

Conclusions:

• A novel, sensitive, and reproducible fluorescence-based assay for N5-CAIR mutase activity has been established.
• This assay is suitable for high-throughput screening of N5-CAIR mutase inhibitors.
• The assay holds potential for identifying new antimicrobial agents targeting microbial purine biosynthesis.