Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, China.
Laboratory Centre of Life Science, College of Life Sciences, Nanjing Agricultural University, Nanjing, China.
Appl Environ Microbiol. 2018 Jul 17;84(15). doi: 10.1128/AEM.00910-18. Print 2018 Aug 1.
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.
5-羟吡啶酸（5HPA）是一种天然吡啶衍生物，可在环境中被微生物降解。然而，5HPA 代谢的生理、生化和遗传基础仍不清楚。在本研究中，从 JQ135 中克隆了一个负责 5HPA 降解的操纵子 ()。HpaM 是一种单成分黄素腺嘌呤二核苷酸（FAD）依赖性单加氧酶，与报道的单加氧酶的同源性仅为 28%至 31%。HpaM 催化 5HPA 的脱羧羟化反应，生成 2,5-二羟基吡啶（2,5DHP）。HpaM 的单加氧酶活性依赖于 FAD 和 NADH。HpaM 对 5HPA 和 NADH 的表观 值分别为 45.4 μM 和 37.8 μM。发现基因 、 和 分别编码 2,5DHP 双加氧酶、-甲酰马来酸脱水酶和马来酸酰胺水解酶；然而，在 JQ135 中，这三个基因并非 5HPA 降解所必需。此外，编码马来酸异构酶的基因 对于 JQ135 中 5HPA、烟酸和吡啶酸的代谢是必需的，表明它可能是吡啶衍生物代谢的关键基因。本研究中鉴定的基因和蛋白质显示了吡啶衍生物降解的新机制。与苯环不同，吡啶环的电子密度不均匀分布会影响位置反应性和与酶的相互作用；例如， 和 氧化比 氧化更困难。羟基化是吡啶衍生物代谢的重要氧化过程。在以前的报道中，吡啶衍生物的 羟基化是由多成分钼依赖性单加氧酶催化的，而 羟基化是由单成分 FAD 依赖性单加氧酶催化的。本研究鉴定了新的单成分 FAD 依赖性单加氧酶 HpaM，它催化 5HPA 的脱羧羟化反应。此外，我们发现编码马来酸异构酶的 基因对于 JQ135 中 5HPA、烟酸和吡啶酸的代谢至关重要。本研究为吡啶衍生物的微生物代谢提供了新的见解。
