N-heterocyclic aromatic compounds (NHACs) are part of common products such as shampoos, dyes, and drugs. If NHACs are consumed, the organisms that consumed these products can suffer from serious negative effects as these are highly polar molecules and readily interact with water making them an emerging environmental threat. However, several species of soil-dwelling bacteria can degrade NHACs and use these molecules for energy. This biodegradation is catalyzed by hydroxylases, monooxygenases, and oxidoreductases. The enzyme 6-hydroxynicotinic acid 3-monooxygenase (NicC) is involved in the oxidative degradation of nicotinic acid in multiple aerobic bacteria species. Therefore, nicotinic acid and NicC can be used as models for exploring the mechanisms by which NHACs are degraded in bacteria.

Using the unliganded Pseudomonas putida NicC crystal structure we were able to use Dock6 to dock small molecule substrate analogs that were promising in binding studies to the putative active site of NicC. Specific substrates that were initially docked were 6-hydroxynicotinic acid (6-HNA) (K_d = 58 ± 12 µM) and 5-chloro-6-hydroxynicotinic acid (5-Cl-6-HNA) (K_d =7 ± 2 µM). The results from this initial docking determined that Tyr215 is an important residue for substrate binding. Currently, we are docking a library of similar molecules from Zinc database to determine structure activity relationships for small molecule binding.

In addition to characterizing nicotinic acid degradation in P. putida using computational methods, other work is focused on understanding NHAC degradation in a similar soil-dwelling bacterium, Bacillus niacini. While in P. putida, NicC converts 6-HNA to 2,5-dihydroxypyridine (2,5-DHP), in B. niacini 6-HNA is converted to 2,6-DHNA (2,6-dihydronicotinic acid) by a NicC-like protein. Here we present homology models of B. niacini NicC that were made using a variety of servers, including SwissModel, the Phyre2 protein fold recognition server, and the CPHmodels-3.2 server. These models were guided by the sequence homology between Pseudomonas and Bacillus NicC. Current work is focused on binding 6-HNA and other substrate analogs to this model. Together this computational approach has provided interesting insights into bacterial NHAC degradation, which will form the basis for future in vitro experiments.

Katherine Hicks

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