A Novel Degradation Mechanism for Pyridine Derivatives in Alcaligenes faecalis JQ135

Abstract

5-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 (), responsible for 5HPA degradation, was cloned from JQ135. HpaM was a monocomponent flavin adenine dinucleotide (FAD)-dependent monooxygenase and shared low identity (only 28 to 31%) with reported monooxygenases. HpaM catalyzed the decarboxylative hydroxylation of 5HPA, generating 2,5-dihydroxypyridine (2,5DHP). The monooxygenase activity of HpaM was FAD and NADH dependent. The apparent values of HpaM for 5HPA and NADH were 45.4 μM and 37.8 μM, respectively. The genes, , and were found to encode 2,5DHP dioxygenase, -formylmaleamic acid deformylase, and maleamate amidohydrolase, respectively; however, the three genes were not essential for 5HPA degradation in JQ135. Furthermore, the gene , which encodes a maleic acid isomerase, was essential for the metabolism of 5HPA, nicotinic acid, and picolinic acid in JQ135, 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. Unlike the benzene ring, the uneven distribution of the electron density of the pyridine ring influences the positional reactivity and interaction with enzymes; e.g., the and oxidations are more difficult than the oxidations. Hydroxylation is an important oxidation process for the pyridine derivative metabolism. In previous reports, the hydroxylations of pyridine derivatives were catalyzed by multicomponent molybdenum-containing monooxygenases, while the hydroxylations were catalyzed by monocomponent FAD-dependent monooxygenases. This study identified the new monocomponent FAD-dependent monooxygenase HpaM that catalyzed the decarboxylative hydroxylation of 5HPA. In addition, we found that the gene coding for maleic acid isomerase was pivotal for the metabolism of 5HPA, nicotinic acid, and picolinic acid in JQ135. This study provides novel insights into the microbial metabolism of pyridine derivatives.

Identification and characterization of a novelpicgene cluster responsible for picolinic acid degradation inAlcaligenes faecalisJQ135

“…However, the pic gene cluster lacks an isomerase gene. Previous study showed that AFA_16520 ( maiA , also named picG in this study) gene was only the maleic acid cis-trans isomerase gene in A. faecalis JQ135 and PicG was responsible for the last step of PA degradation (25). Furthermore, a DNA fragment containing picEDFG genes was cloned into a 2,5DHP non-degrading bacterium Sphingomonas wittichii DC-6 (35).…”

“…In A. faecalis JQ135, the 3,6DHPA was found to be degraded into 2,5DHP by 3,6-dihydroxypicolinic acid decarboxylase (PicC) (26). 2,5DHP is a central intermediate of numerous pyridine derivatives, including nicotinate (28), nicotine (33), 2-hydroxy-pyridine (34), and 5-hydroxypicolinic acid (25). The bacterial metabolism of 2,5DHP usually employs the maleamate pathway, whose key enzymes include 2,5DHP dioxygenase, N -formylmaleamate (NFM) deformylase, maleamate amidase, and maleate cis-trans isomerase (28, 33).…”

“…All bacterial strains and plasmids used in this study are listed in Table 2. Mineral salts medium (MSM) and Luria-Bertani medium (LB) have been described previously (25). Alcaligenes faecalis JQ135 was the wild-type PA-degrading strain (24).…”

“…For the over-expression of picD gene in E. coli BL21(DE3), the complete ORF without its corresponding stop codon was amplified and inserted into the NdeI/XhoI-digested plasmid pET29a(+), resulting in the plasmid pET-PicD. The inductuion and purification of 6×His-tagged PicD from E. coli BL21(DE3) (containing pET-PicD) were same with a previous report (25). The purified 6×His-tagged PicD were then analyzed by 12.5% SDS-PAGE.…”

“…Alcaligenes faecalis strain JQ135 can degrade PA and utilize it for cell growth (24) (25). Our previous study demonstrated the first genetic evidence involving PA degradation, in which picC (encoding 3,6-dihydroxypicolinic acid decarboxylase) gene was responsible for the conversion of 3,6DHPA to 2,5DHP in A. faecalis strain JQ135 (26).…”