The enzyme N -carboxylaminoinidazole ribonucleotide (N -CAIR) mutase is found in microbial de novo purine biosynthesis but is absent in humans making it an attractive antimicrobial target. N -CAIR mutase catalyzes the synthesis of carboxyaminoimidazole ribonucleotide (CAIR) from N -CAIR which is itself prepared from aminoimidazole ribonucleotide (AIR) by the enzyme N -CAIR synthetase. During our research on identifying inhibitors of N -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 N -CAIR mutase.
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Carboxylation in de novo purine biosynthesis.
Methods Enzymol
November 2024
Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI, United States. Electronic address:
Article Synopsis
- De novo purine biosynthesis is crucial for producing purine nucleotides necessary for biological functions like nucleic acid replication, involving a key reaction where 5-aminoimidazole ribonucleotide (AIR) is carboxylated to form 4-carboxy-5-aminoimidazole ribonucleotide (CAIR).
- This carboxylation occurs differently across species: humans and higher eukaryotes use AIR carboxylase to directly synthesize CAIR, while bacteria, yeast, fungi, and plants do it in two steps involving N-CAIR synthetase and N-CAIR mutase.
- Understanding these divergent pathways has implications for developing antimicrobial agents and targeting related enzymes in cancer treatment
Although purine biosynthesis is a primary metabolic pathway, there are fundamental differences between how purines are synthesized in microbes versus humans. In humans, the purine intermediate, 4- carboxy-5-aminoimidazole ribonucleotide (CAIR) is directly synthesized from 5-aminoimidazole ribonucleotide (AIR) and carbon dioxide by the enzyme AIR carboxylase. In bacteria, yeast and fungi, CAIR is synthesized from AIR via an intermediate N-carboxyaminoimidazole ribonucleotide (N-CAIR) by the enzyme N-CAIR mutase.
View Article and Find Full Text PDFStructure-Guided Discovery of N-CAIR Mutase Inhibitors.
Biochemistry
September 2023
The Hormel Institute, University of Minnesota, Austin, Minnesota 55912, United States.
Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of antimicrobial therapies. While humans biosynthesize purine nucleotides in a 10-step pathway, most microbes utilize an additional 11th enzymatic activity. The human enzyme, aminoimidazole ribonucleotide (AIR) carboxylase generates the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) directly.
View Article and Find Full Text PDFA Fluorescence-Based Assay for N -Carboxyaminoimidazole Ribonucleotide Mutase.
Chembiochem
September 2023
Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Avenue, 48201, Detroit, MI, USA.
The enzyme N -carboxylaminoinidazole ribonucleotide (N -CAIR) mutase is found in microbial de novo purine biosynthesis but is absent in humans making it an attractive antimicrobial target. N -CAIR mutase catalyzes the synthesis of carboxyaminoimidazole ribonucleotide (CAIR) from N -CAIR which is itself prepared from aminoimidazole ribonucleotide (AIR) by the enzyme N -CAIR synthetase. During our research on identifying inhibitors of N -CAIR mutase, we developed an innovative, fluorescence-based assay to measure the activity of this enzyme.
View Article and Find Full Text PDFIsatins Inhibit N-CAIR Synthetase by a Substrate Depletion Mechanism.
Biochemistry
April 2019
Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences , Wayne State University, Detroit , Michigan 48201 , United States.
The continued rise of antibiotic-resistant infections coupled with the limited pipeline of new antimicrobials highlights the pressing need for the development of new antibacterial agents. One potential pathway for new agents is de novo purine biosynthesis as studies have shown that bacteria and lower eukaryotes synthesize purines differently than humans. Microorganisms utilize two enzymes, N-CAIR synthetase and N-CAIR mutase, to convert 5-aminoimidazole ribonucleotide (AIR) into 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) through the intermediate N-carboxy-5-aminoimidazole ribonucleotide (N-CAIR).
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