Biophysical Society Thematic Meeting | Trieste 2024

Emerging Theoretical Approaches to Complement Single-Particle Cryo-EM

Poster Abstracts

32-POS Board 32 DECIPHERING PURINE METABOLIC REGULATION IN E. COLI ADENINE PHOSPHORIBOSYLTRANSFERASE (APRT): A MULTI-METHOD APPROACH Priya Yadav 1 ; Gajraj Singh Kushwaha 1 ; Neel Sarovar Bhavesh 1 ; 1 International Centre for Genetic Engineering and Biotechnology, (ICGEB), New Delhi, Integrative Biology, New Delhi, India Nucleoside analogues have been invaluable in treating cancer, viral, and fungal diseases. However, the challenges of multidrug resistance, dormancy, and latency in bacterial treatments have prompted their screening as antibacterial targets in nucleotide metabolism. The salvage pathway of nucleotide synthesis provides an energy-efficient supply of nucleotides, maintaining cellular pools, adapting to nutrient-starved conditions, and ensuring antibiotic tolerance. Adenine phosphoribosyltransferase (APRT), salvage pathway enzyme, catalyzes a reversible reaction converting adenine into adenosine monophosphate (AMP). In proteobacteria, a large class of human pathogenic bacteria, the mechanistic regulation of APRT remains underexplored. This study aims to decipher the regulation of purine metabolism by APRT in this class and design potential inhibitors.We determined atomic-resolution structures of the apo and substrate-bound (adenine, AMP) forms of E. coli APRT using X-ray crystallography, revealing unique mechanistic regulations of substrate binding within the active site. Our crystallographic and isothermal calorimetry data concluded a new mode of adenine binding which is PRPP independent binding of adenine in the active site, contrary to the other APRTs which are well known PRPP-dependent binding of adenine. The catalytic residues which we identified in enzyme assay are 14 Å away from the nucleobase ring in determined monosubstrate bound forms. Therefore, we are employing a scaffold-based cryo-EM approach to capture a transition state where both adenine and PRPP are bound. The cryo-EM data, analyzed with MD simulations and existing crystallographic, enzymatic, and thermodynamic data, will uncover metastable and transition states in proteobacterial APRT activity. Targeting these states with inhibitors offers a promising strategy for antimicrobial interventions.

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