Determining the mechanism of 6‐hydroxynicotinate 3‐monooxygenase (NicC), an enzyme involved in nicotinate degradation

Abstract

N‐Heterocyclic aromatic compounds (NHACs) are employed in daily life and are environmental contaminants. Due to NHAC's toxic, mutagenic, and carcinogenic effects, improved purification methods are imperative. Understanding bacterial degradation of NHACs may assist future bioremediation techniques. Nicotinic acid (NA) degradation serves as a good model for NHAC catabolism. 6‐Hydroxynicotinate‐3‐monooxygenase (NicC) is an enzyme involved in the degradation of NA. NicC catalyzes the hydroxylative decarboxylation of 6‐hydroxynicotinic acid (6‐HNA) and NADH to 2,5‐dihydroxypyridine (2,5‐DHP) and NAD. Understanding the mechanism of this reaction, which activates the pyridine of NA for ring‐opening, will provide greater insight on the conditions NHAC degradation require in bacteria. This knowledge could result in the optimization of bioremediation efforts that are already known and reduce the amount of hazardous waste in the environment. Two mechanisms for the oxidation half‐reaction of NicC have been proposed. One mechanism (A) predicts the formation of a covalent complex between NicC and 6‐HNA, decarboxylation, and subsequent hydroxylation by hydroperoxyflavin. A second mechanism (B) proposes a base deprotonates 6‐HNA leading to hydroxylation by hydroperoxyflavin and then decarboxylation. The research objective is to elucidate the mechanism of this reaction. Current results suggest NicC catalyzes oxidative decarboxylation by mechanism B. ESI‐MS data indicate no evidence of a covalent complex between the enzyme and the substrate, as the protein mass was equivalent to only the enzyme and not the ES complex. Mechanism A involves the keto tautomer requiring nitrogen to be present in the aromatic ring, while mechanism B has no such requirement. To test this, NicC was shown to catalyze the reaction of 4HBA, a homocyclic analog of 6‐HNA. This result confirms that NicC doesn't require nitrogen in the ring and disproves mechanism A. The coupling of the WT NicC reaction was determined to generate one NAD for every 2,5‐DHP with a time point HPLC assay. Future work will compare the coupling and rates of the WT NicC reaction to those of the variant NicC reactions to identify the key catalytic residues.Support or Funding InformationHenry J. Copeland Fund for Independent Study at the College of WoosterThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.