ABSTRACT5-Hydroxypicolinic acid (5HPA), a natural pyridine derivative, is microbially degraded in the environment. However, the physiological, biochemical, and genetic foundations of 5HPA metabolism remain unknown. In this study, an operon (hpa), responsible for 5HPA degradation, was cloned fromAlcaligenes faecalisJQ135. HpaM was a monocomponent flavin adenine dinucleotide (FAD)-dependent monooxygenase and shared low identity (only 28 to 31%) with reported monooxygenases. HpaM catalyzed theorthodecarboxylative hydroxylation of 5HPA, generating 2,5-dihydroxypyridine (2,5DHP). The monooxygenase activity of HpaM was FAD and NADH dependent. The apparentKmvalues of HpaM for 5HPA and NADH were 45.4 μM and 37.8 μM, respectively. The geneshpaX,hpaD, andhpaFwere found to encode 2,5DHP dioxygenase,N-formylmaleamic acid deformylase, and maleamate amidohydrolase, respectively; however, the three genes were not essential for 5HPA degradation inA. faecalisJQ135. Furthermore, the genemaiA, which encodes a maleic acidcis-transisomerase, was essential for the metabolism of 5HPA, nicotinic acid, and picolinic acid inA. faecalisJQ135, indicating that it might be a key gene in the metabolism of pyridine derivatives. The genes and proteins identified in this study showed a novel degradation mechanism of pyridine derivatives.IMPORTANCEUnlike the benzene ring, the uneven distribution of the electron density of the pyridine ring influences the positional reactivity and interaction with enzymes; e.g., theorthoandparaoxidations are more difficult than themetaoxidations. Hydroxylation is an important oxidation process for the pyridine derivative metabolism. In previous reports, theorthohydroxylations of pyridine derivatives were catalyzed by multicomponent molybdenum-containing monooxygenases, while themetahydroxylations were catalyzed by monocomponent FAD-dependent monooxygenases. This study identified the new monocomponent FAD-dependent monooxygenase HpaM that catalyzed theorthodecarboxylative hydroxylation of 5HPA. In addition, we found that themaiAgene coding for maleic acidcis-transisomerase was pivotal for the metabolism of 5HPA, nicotinic acid, and picolinic acid inA. faecalisJQ135. This study provides novel insights into the microbial metabolism of pyridine derivatives.