Aqlesia Aregawi | 2025 I.S Symposium

Name: Aqlesia Aregawi
Title: Investigating Substrate Promiscuity of 6-Hydroxynicotinate 3-monooxygenase (NicC): NicC Activity with 5-Chloro-Coumalate
Major: Biochemistry & Molecular Biology
Advisors: Mark J. Snider

6-Hydroxynicotinate 3-monooxygenase (NicC) catalyzes the decarboxylative hydroxylation of 6-HNA, a key intermediate involved in the degradation of nicotinic acid by aerobic bacteria. As a N-heterocyclic aromatic compound, the biodegradation of nicotinic acid serves as a model for exploring the mechanisms and specificity of critical enzymes in the biodegradation of environmental pollutants. Earlier studies of NicC have demonstrated its ability to bind and catalyze the hydroxylation and decarboxylation of chlorinated substrate mimics. This study aims to better understand substrate promiscuity of NicC by investigating how chlorination of a poor substrate, coumalate, in which the pyridine ring’s N atom of 6-HNA has been replaced with O, affects its activity with NicC. LC-MS analysis confirmed that NicC catalyzes the formation of 3-Cl-2,5-dihydroxy pyran-one with m/z of 144.97. The HPLC analysis of resulting product also revealed that the reaction becomes uncoupled, with approximately 19 ± 15% of the NADH oxidation resulting in hydrogen peroxide instead 3-Cl-2,5-dihydroxy pyran-one formation. Steady state kinetic analysis indicates that the catalytic efficiency of NicC with 5-Cl-coumalate (kcat/KM = (3.9 ± 1.3) ×104 M-1s-1) is better than coumalate (kcat/KM = 6.4 x 103 M-1s-1). Transient state kinetics of the reductive half-reaction by stopped flow spectrophotometry revealed that the Kd (of NADH) = 39 ± 24 micromolar and maximum reduction rate constant of 3.5 s-1. In comparison to the reduction kinetics with coumalate (Kd of NADH = 670 ± 27 micromolar), the chlorination causes a drop in Kd value of NADH. The Kd of NADH with 6-HNA (3 micromolar) is 13× lower than 5-Cl-coumalate with a reduction rate constant (kred = 44 s-1) that is 12× quicker. These results are consistent with past observations that chlorination of the substrate enhances the catalytic efficiency of NicC and demonstrates how chlorination allows NicC to act on what was otherwise a very poor substrate.

Posted in Symposium 2025 on May 1, 2025.