Determination and Characterization of Genes that Encode the Nicotinate Dehydrogenase and 6‐Hydroxynicotinate Dehydrogenase Complexes within the Nicotinic Acid Degradation Pathway by Bacillus Niacini

Abstract

N‐Heterocyclic aromatic compounds (NHACs) are constituents of many manufactured products such as pharmaceuticals, vitamins, and personal care products. Their widespread use has led to contamination of groundwater and soils, posing environmental concerns. Therefore, mechanisms of how NHACs are catabolized by bacteria have been studied to develop bioremediation strategies. Nicotinic acid (NA) is an example of an NHAC where its catabolism has been studied in a variety of microorganisms, such as Pseudomonas, Eubacterium,and Bacillus. While NA degradation has been elucidated in microorganisms such as P. putidaand E. barkeri,the majority of B. niacini’sNA catabolism pathway is relatively uncharacterized. Previous work identified a nicgene cluster that had been found to be upregulated in the presence of NA and 6‐hydroxynicotinic acid (6‐HNA), leading to the hypothesis that the set of protein subunits NicA1, NicA2, NicB1, NicB2 and CoxG are involved in the first two hydroxylation steps of B. niacini’sNA degradation pathway. These proteins are thought to form distinct multi‐subunit protein complexes with nicotinate dehydrogenase (NDH) and 6‐hydroxynicotinate dehydrogenase (6‐HNDH) activities. Due to the involvement of the molybdopterin cofactor present in the B subunits, recombinant forms of these enzymes have not shown activity when expressed and purified from E. coli. A vector specific for expressing the genes in Pseudomonas (pBBr) was engineered to contain the set of five B. niacini genes hypothesized to code for NA hydroxylating enzymes and was transformed into Pseudomonas fluorescens 1f2,a strain shown to be unable to degrade nicotinic acid naturally. Analysis of the media upon incubation of the Pf‐1f2 transformants with NA or 6‐HNA in minimal media by C‐18 chromatography (HPLC) showed rapid and complete hydroxylation of NA to 6‐HNA, as well as the hydroxylation of 6‐HNA to 2,6‐dihydroxynicotinic acid (2,6‐DHNA). This is the first evidence of activity observed from recombinant B. niacini“NicAB” protein complexes. However, the combination of gene‐encoded subunits that compose the NDH and 6‐HNDH complexes is currently unknown. Using deletion mutagenesis, specific genes cloned into the pBBr vector will be removed to recombinantly express variant complexes that lack a specific subunit to determine the substrate specificity and oligomeric structure of the complexes.