Novel Molecular Insights into Isonicotinic Acid Biodegradation by <i>Alicycliphilus denitrificans</i> Strain DP3: Genomics Characterization, Molecular Mechanisms, and Pathways

Isonicotinic acid (INA) is a widely used chemical and agricultural intermediate, which contaminates agricultural products, causes lysine isonicotinylation of histones, and increases the risk of cancer in humans. The microbial degradation of INA has been continuously investigated for 60 years, but its molecular mechanism remains unclear. In this study, a newly isolated strain, <i>Alicycliphilus denitrificans</i> DP3, was found to degrade 2.5 mM INA to undetectable levels within 48 h. A novel <i>ina</i> gene cluster located on the plasmid encodes two consecutive three-component molybdenum-dependent hydroxylases: InaA1A2A3 catalyzes the first hydroxylation of INA to 2-hydroxyisonicotinate (2HINA), while InaB1B2B3 catalyzes the second hydroxylation to 2,6-dihydroxyisonicotinate (26HINA). H₂¹⁸O isotope labeling confirmed that the incorporated oxygen atoms are derived from water. RT-qPCR demonstrated that seven <i>ina</i> genes were strongly induced by INA. Comparative genomics showed that homologous <i>ina</i> clusters are globally distributed, and these findings provide the first genetic framework for microbial INA degradation.