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Elucidating The Nicotinic Acid Degradation Pathway In Bacillus niacini: Identification and Biochemical Characterization Of Proteins Of Unknown Function

N‐Heterocyclic aromatic compounds (NHACs) are abundant in the manufacturing of solvents, pesticides, and dyes; however, accumulation of these compounds can lead to detrimental environmental impacts. To alleviate these impacts, it is critical to understand the mechanisms microbial species use to catabolize these types of molecules so that bioremediation techniques can be implemented. Nicotinic acid is commonly used as a model system for NHAC degradation because it is environmentally safe and can be degraded by a variety of bacterial species. Bacillus niacini is a soil‐dwelling bacterium that has been identified to degrade NA through an uncharacterized pathway. This makes B. niacini an organism of interest, as elucidation of the mechanism by which it catabolizes NA will reveal differences between these pathways that can be exploited to optimize bioremediation efforts. Bioinformatic analyses of genomic and transcriptomic data revealed a potential operon responsible for NA degradation in B. niacini. This operon contains 15 genes that encode for 7 enzymes that are necessary for NA degradation as well as three genes that have not yet been elucidated: a domain of unknown function (DUF), a flavin monooxygenase (FMO), and a hypothetical protein (HP). NA degradation in B. niacini is unique in that it involves two ring hydroxylating steps, forming the intermediate 2,6‐dihydroxynicotinic acid (DHNA). After decarboxylation of DHNA to 2,6‐dihydroxypridine (DHP), we show that the DUF hydroxylates DHP to 2,3,6‐trihydroxypyridine (THP). Separation of the DUF‐catalyzed reaction products by C‐18 chromatography (HPLC) and detection by UV‐Vis at pH 3 shows a new species with an Absmaxat 320 nm and 590 nm. Mass spectrometry (Q‐TOF) analysis of the reaction identifies the products as DHP‐ and THP‐ oxidized dimers (nicotine blue). Formation of these products are observed to be O2 dependent. When the DUF, FMO and HP genes are recombinantly expressed together in E. coli, the cell extract is able to degrade DHP without forming the DHP‐oxidized dimer. That observation suggests the possible roles of FMO and/or HP in catalyzing the conversion of the DHP‐dimer back to DHP. This study is the first to characterize the genes responsible for NA degradation by B. niacini and further establishes the NA degradation pathway.