Jack Donahue | 2024 I.S. Symposium
Name: Jack Donahue
Title: Role of Residues R184 and W273 in 6-HNA and NADH Binding by 6-Hydroxynicotinate 3-Monooxygenase
Major: Biochemistry and Molecular Biology
Advisor: Mark Snider; James West (second reader)
Industrial pollution has led to pervasive environmental contamination by N-heterocyclic aromatic compounds (NHACs). Fortunately, soil bacteria degrade these compounds into common metabolic intermediates. Aerobic degradation of the NHAC nicotinic acid involves hydroxylating then opening the ring, a common theme in NHAC degradation. The class A flavin monooxygenase 6-hydroxynicotinate 3-monooxygenase (NicC) uses an FAD coenzyme and NADH oxidation to catalyze the second step of nicotinic acid degradation, a decarboxylative hydroxylation reaction from 6-HNA to 2,5-DHP. While the binding sites for FAD and 6-HNA are understood, the NADH binding site in NicC is unknown. The goal of this study is to uncover the binding site of NADH to NicC in relation to 6-HNA binding. Previous research suggested the FAD binding site and a secondary tunnel as possible NADH binding sites. The residues of interest in this study are R184 and W273, located in the active site, and R278, on the exterior of the enzyme. Pseudomonas putida NicC (PpNicC) variants R184K, W273Y, and R278E were engineered and purified from E. coli. Steady-state kinetic analysis by method of initial rates was performed and Michaelis-Menten plots were constructed for each variant as a function of 6-HNA or NADH. By comparison to WT PpNicC, it was established that W273Y and R184K decreased the affinity of NicC for 6-HNA without changing catalytic turnover. Further, it was determined that R278E and W273Y moderately decreased NADH affinity whereas R184K decreased NADH affinity 300-fold. This suggests NADH binds in the FAD site of the enzyme and residue R184 is critical for this association. This leads to the development of a novel hypothesis that R184 functions to control FAD conformation dynamics upon 6-HNA binding. This study gives a better understanding of catalysis in NicC, confirms past findings, and introduces R184 as a residue of interest for stopped-flow catalysis work to illustrate its impacts on NADH binding.
Posted in Symposium 2024 on May 2, 2024.
