Enzymatic characterization and structural analysis of flavin monooxygenases involved in bacterial nicotinic acid degradation

Creator

Savannah Brancato, SUNY Cortland
Emmalee Cooke, SUNY Cortland
Jonathan Gwilt, SUNY Cortland

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Faculty Mentor

Katherine Hicks

Creation Date

5-2020

Description

N-heterocyclic aromatic compounds, or NHACs, are commonly found in shampoos, dyes, pharmaceuticals, industrial solvents and other anthropogenic products. Due to their polarity, these compounds are highly water soluble and can be toxic to both aquatic and soil-dwelling organisms. NHACs are also known to serve as a source of carbon biomass for certain bacteria, and several metabolic enzymes are involved in their stepwise breakdown. Understanding the structures and chemical mechanisms of the bacterial enzymes involved in the degradation of NHACs will allow for more efficient bioremediation efforts.

Both Bacillus niacini and Pseudomonas putida have pathways which degrade the NHAC nicotinic acid (NA), leading to the formation of the less toxic metabolite, fumaric acid. While both bacteria act on the same initial NA substrate, the intermediates and enzymes utilized along the pathways vary. Specifically, the P. putida pathway involves a six-step process, the second of which involves the enzyme 6-hydroxynicotinic acid 3-monooxygenase (NicC). NicC catalyzes the decarboxylative hydroxylation of 6-hydroxynicotinic acid (6-HNA) to 2,5-dihydroxy pyridine (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). Specifically, the analogs 5-chloro 6-hydroxy nicotinic acid (K_d= 7 ± 2 μM) and coumalic acid (K_d= 6 ± 1 μM) were chosen based on their high binding affinity relative to the natural 6-HNA substrate (K_d= 85 ± 13 μM) and low catalytic turnover in the absence of NADH.

A similar step in the B. niacini NA metabolic pathway involves the conversion of 2,6-dihydroxy nicotinic acid (2,6-DHNA) to 2,6-dihydroxypyridine (2,6-DHP) by a flavin monooxygenase (FMO) that is currently structurally and mechanistically uncharacterized. Here we present preliminary results of crystallization experiments on the N-terminally tagged-His₆ protein which resulted in low quality crystals. However, these preliminary leads are guiding current efforts to crystallize the untagged FMO protein. We are also working to synthesize potential FMO substrates and obtain a liganded NicC structure. Together these data will provide insights into the catalytic mechanisms of these enzymes, and aid in their potential utilization in bioremediation efforts.