Mayank Raj Pandey | 2025 I.S Symposium

Name: Mayank Raj Pandey
Title: Investigating the Importance of Arg 108 in the Binding of NADH by 6-Hydroxynicotinate 3-monooxygenase (nicC)
Major: Biochemistry & Molecular Biology
Advisors: Mark J. Snider

Nicotinic acid (NA), a carboxylic derivative of pyridine, represents a model substrate in microbial degradation pathways of N-heterocyclic aromatic compounds (N-HACs), environmental pollutants with carcinogenic and toxic properties. 6-Hydroxynicotinate 3-monooxygenase (NicC) catalyzes the conversion of 6-hydroxynicotinate (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP), a pivotal step in NA degradation requiring NADH-mediated reduction of flavin adenine dinucleotide (FAD). Despite the critical role of NADH in NicC’s enzymatic activity, the amino acid determinants of its binding remain uncharacterized. This study focuses on Arg 108, a conserved residue hypothesized to stabilize NADH binding. Phylogenetic analysis of NicC orthologs reveals that Arg 108 is highly conserved across bacterial species, underscoring its functional significance. Structural modeling and docking studies suggest that Arg 108 facilitates NADH stabilization via H-bonding interactions, which was further investigated through site-directed mutagenesis. The heterologously expressed and purified R108K NicC variant exhibited a higher Km and reduced catalytic efficiency, in comparison to WT NicC, as determined by steady-state kinetic analyses. These findings implicate Arg 108 as a potential key residue in NicC’s catalytic mechanism, which affects effective hydride transfer to FAD. This research aims to elucidate the molecular basis of NADH binding in NicC, offering insights into the enzyme’s role in N-HAC biodegradation and providing insight into the requirements of NicC substrate specificity.

Posted in Symposium 2025 on May 1, 2025.