Picolinic acid (PA), a typical C-2-carboxylated pyridine derivative, is a metabolite of l-tryptophan and many other aromatic compounds in mammalian and microbial cells. Microorganisms can degrade and utilize PA for growth. However, the precise mechanism of PA metabolism remains unknown. Alcaligenes faecalis strain JQ135 utilizes PA as its carbon and nitrogen source for growth. In this study, we screened a 6-hydroxypicolinic acid (6HPA) degradation-deficient mutant through random transposon mutagenesis. The mutant hydroxylated 6HPA into an intermediate, identified as 3,6-dihydroxypicolinic acid (3,6DHPA), with no further degradation. A novel decarboxylase, PicC, was identified to be responsible for the decarboxylation of 3,6DHPA to 2,5-dihydroxypyridine. Although, PicC belonged to the amidohydrolase 2 family, it shows low similarity (<45%) compared to other reported amidohydrolase 2 family decarboxylases. Moreover, PicC was found to form a monophyletic group in the phylogenetic tree constructed using PicC and related proteins. Further, the genetic deletion and complementation results demonstrated that picC was essential for PA degradation. The PicC was Zn2+-dependent nonoxidative decarboxylase that can specifically catalyze the irreversible decarboxylation of 3,6DHPA to 2,5-dihydroxypyridine. The Km and k cat toward 3,6DHPA were observed to be 13.44 μM and 4.77 s-1, respectively. Site-directed mutagenesis showed that His163 and His216 were essential for PicC activity. This study provides new insights into the microbial metabolism of PA at molecular level.IMPORTANCE Picolinic acid is a natural toxic pyridine derived from l-tryptophan metabolism and other aromatic compounds in mammalian and microbial cells. Microorganisms can degrade and utilize picolinic acid for their growth, and thus a microbial degradation pathway of picolinic acid has been proposed. Picolinic acid is converted into 6-hydroxypicolinic acid, 3,6-dihydroxypicolinic acid, and 2,5-dihydroxypyridine in turn. However, there was no physiological and genetic validation for this pathway. This study demonstrated that 3,6-dihydroxypicolinic acid was an intermediate in picolinic acid catabolism and further identified and characterized a novel amidohydrolase 2 family decarboxylase PicC. PicC was also shown to catalyze the decarboxylation of 3,6-dihydroxypicolinic acid into 2,5-dihydroxypyridine. This study provides a basis for understanding picolinic acid degradation and its underlying molecular mechanism.

中文翻译：

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粪产碱菌JQ135中新型3,6-二羟基吡啶甲酸脱羧酶介导的吡啶甲酸分解代谢。

吡啶甲酸（PA）是一种典型的C-2-羧化吡啶衍生物，是l-色氨酸和许多其他芳香族化合物在哺乳动物和微生物细胞中的代谢物。微生物可以降解并利用PA进行生长。但是，PA代谢的确切机制仍然未知。产碱杆菌粪便菌株JQ135利用PA作为其生长的碳源和氮源。在这项研究中，我们通过随机转座子诱变筛选了6-羟基吡啶甲酸（6HPA）降解缺陷的突变体。突变体将6HPA羟基化为中间体，称为3,6-二羟基吡啶甲酸（3,6DHPA），没有进一步降解。一种新型的脱羧酶PicC被认为是3,6DHPA脱羧成2,5-二羟基吡啶的原因。尽管PicC属于酰胺水解酶2家族，但其相似性较低（< 与其他报道的酰胺水解酶2家族的脱羧酶相比，降低了45％）。此外，发现在使用PicC和相关蛋白构建的系统发育树中，PicC形成了一个单系群。此外，遗传删除和互补结果表明picC对于PA降解至关重要。PicC是依赖Zn2 +的非氧化脱羧酶，可特异性催化3,6DHPA不可逆地脱羧为2,5-二羟基吡啶。观察到朝向3,6DHPA的Km和k cat分别为13.44μM和4.77 s-1。定点诱变表明，His163和His216对PicC活性至关重要。这项研究提供了在分子水平上PA微生物代谢的新见解。重要信息吡啶甲酸是一种天然的有毒吡啶，来源于哺乳动物和微生物细胞中的色氨酸代谢和其他芳香族化合物。微生物可以降解并利用吡啶甲酸来促进其生长，因此提出了吡啶甲酸的微生物降解途径。吡啶甲酸依次转化为6-羟基吡啶甲酸，3,6-二羟基吡啶甲酸和2,5-二羟基吡啶。但是，该途径尚无生理和遗传学验证。这项研究表明3,6-二羟基吡啶甲酸是吡啶甲酸分解代谢的中间体，并进一步鉴定和表征了一种新型的酰胺水解酶2家族脱羧酶PicC。PicC还显示可催化3,6-二羟基吡啶甲酸脱羧为2,5-二羟基吡啶。