The Novel Monocomponent FAD-dependent Monooxygenase HpaM Catalyzes the 2-Decarboxylative Hydroxylation of 5-Hydroxypicolinic Acid in Alcaligenes faecalis JQ135

5-hydroxypicolinic acid (5HPA) is a natural pyridine derivative that can be microbially degraded. However, the physiological, biochemical, and genetic foundation of the microbial catabolism of 5HPA remains unknown. In this study, a gene cluster hpa (which is involved in degradation of 5HPA in Alcaligenes faecalis JQ135) was cloned and HpaM was identified as a novel monocomponent FAD-dependent monooxygenase. HpaM shared a sequence only 31% similarity with the most related protein 6-hydroxynicotinate 3-monooxygenase (NicC) of Pseudomonas putida KT2440. hpaM was heterologously expressed in E. coli BL21(DE3), and the recombinant HpaM was purified via Ni-affinity chromatography. HpaM catalyzed the 2-decarboxylative hydroxylation of 5-HPA, thus generating 2,5-dihydroxypyridine (2,5-DPH). Monooxygenase activity was only detected in the presence of FAD and NADH, but not of FMN and NADPH. The apparent Km values of HpaM toward 5HPA and NADH were 45.4 μM and 37.8 μM, respectively. Results of gene deletion and complementation showed that hpaM was essential for 5HPA degradation in Alcaligenes faecalis JQ135.