Deciphering the Metal Dependency of Novel Nicotinate Hydroxylase from Bacillus niacini

N ‐Heterocyclic compounds are becoming increasingly common in the environment due to their miscibility in water and pose potential threats to wildlife due to their carcinogenic properties. Nicotinic acid (NA), a nontoxic ‐ N‐ heterocyclic aromatic compound, serves as a useful model for this class of compounds and is known to be degraded by a number of soil bacteria. Knowledge of the chemistries involved in NA degradation by these bacteria can be applied to bioremediation techniques to remove similar contaminates from the environment. Bacillus niacini is a common soil bacterium that is able to completely degrade and utilize nicotinic acid by a pathway that is largely uncharacterized. A gene cluster hypothesized to code the enzymes responsible for nicotinate degradation in B. niacini suggests that four proteins are involved in the first two steps of the nicotinate degradation pathway. The sequences predict that two Mo‐dependent hydroxylases (NicB 1 and NicB 2 ), a flavin‐dependent protein (NicA 2 ) and a protein containing an [2Fe‐2S] cluster (NicA 1 ) are potentially important for the first two oxidation steps of nicotinate degradation. To determine the metal dependency and further understand the mechanism of the NicAB enzymes, heterologous expression and purification of each of these enzymes was achieved from E . coli and Pseudomonas chlororaphis, analyzed for metal content and ability to catalyze the double hydroxylation of nicotinate to 2,6‐dihydroxynicotinate. Support or Funding Information This work was supported by funding from the College of Wooster Department of Chemistry and the National Science Foundation.