N-heterocyclic aromatic compounds (NHACs) are environmental toxins commonly found in shampoos, dyes, pharmaceuticals, and other anthropogenic products. The high polarity of these compounds makes them highly soluble in water, thereby posing a threat to aquatic and soil-dwelling organisms. However, NHACs can serve as a source of carbon biomass for bacteria in which several metabolic enzymes are involved in the gradual breakdown of these toxic compounds. Understanding the molecular structures and chemical mechanisms of these bacterial enzymes will lead to more efficient and successful bioremediation efforts. The bacteria strains Bacillus niacini and Pseudomonas putida each have metabolic pathways that degrade the NHAC nicotinic acid (NA) to a less toxic metabolite known as fumaric acid. Although both bacteria strains act on the same initial NA substrate, the intermediates and enzymes the bacteria use in their metabolic pathway vary. Specifically, the P. putida pathway involves a six-step process, and the second enzyme in this pathway is a 6-hydroxynicotinic acid 3-monooxygenase (NicC). NicC catalyzes the decarboxylative hydroxylation of 6-hydroxynicotinic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP). To further understand the catalytic mechanism of NicC, purified protein was crystallized in the presence of substrate analogs in an attempt to attain ligand-bound structure(s). These experiments resulted in non-diffracting crystals, so current trials involve purifying NicC in the absence of any halides, which are hypothesized to inhibit substrate binding in similar enzymes, prior to crystallization experiments. In the B. niacini NA metabolic pathway, there is a similar decarboxylative hydroxylation step that involves the conversion of either 2,6-dihydroxynicotinic acid (2,6-DHNA) to 2,6-dihydroxypyridine (2,6-DHP) or 2,6 dihydroxypyridine (2,6-DHP) to 2,6-DHP. This step involves a flavin monooxygenase (FMO) enzyme that is currently structurally and mechanistically uncharacterized. Using 13C and 1H NMR spectrometry, the ability of 2,6-DHNA to spontaneous decarboxylate will be measured to ascertain whether 2,6-DHNA or 2,6-DHP is the likely substrate for the B. niacini FMO enzyme. Kinetic assays will also be performed a UV-visible spectrophotometric assay with both 2,6-DHNA and 2,6-DHP. Together these data will provide insights into the catalytic mechanisms of these enzymes, and aid in their potential utilization in bioremediation efforts.

Biochemical and structural analysis of bacterial flavin monooxygenases involved in nicotinic acid metabolism

ACS Fall 2020 Virtual Meeting

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