Abstract：
In various organisms, the coenzyme form of vitamin B6, pyridoxal phosphate (PLP), is synthesized from pyridoxine phosphate (PNP). Control of PNP levels is crucial for metabolic homeostasis because PNP has the potential to inhibit PLP-dependent enzymes and proteins. Although the only known pathway for PNP metabolism in Escherichia coli involves oxidation by PNP oxidase, we detected a strong PNP phosphatase activity in E. coli cell lysate. To identify the unknown PNP phosphatase(s), we performed a multicopy suppressor screening using the E. coli serA pdxH strain, which displays PNP-dependent conditional lethality. The results showed that overexpression of the yigL gene, encoding a putative sugar phosphatase, effectively alleviated the PNP toxicity. Biochemical analysis revealed that YigL has strong phosphatase activity against PNP. A yigL mutant exhibited decreased PNP phosphatase activity, elevated intracellular PNP concentrations, and increased PNP sensitivity, highlighting the important role of YigL in PNP homeostasis. YigL also shows reactivity with PLP. The phosphatase activity of PLP in E. coli cell lysate was significantly reduced by mutation of yigL and nearly abolished by additional mutation of ybhA, which encodes putative PLP phosphatase. These results underscore the important contribution of YigL, in combination with YbhA, as a primary enzyme in the dephosphorylation of both PNP and PLP in E. coli.IMPORTANCEPyridoxine phosphate (PNP) metabolism is critical for both vitamin B6 homeostasis and cellular metabolism. In Escherichia coli, oxidation of PNP was the only known mechanism for controlling PNP levels. This study uncovered a novel phosphatase-mediated mechanism for PNP homeostasis. Multicopy suppressor screening, kinetic analysis of the enzyme, and knockout/overexpression studies identified YigL as a key PNP phosphatase that contributes to PNP homeostasis when facing elevated PNP concentrations in E. coli. This study also revealed a significant contribution of YigL, in combination with YbhA, to PLP metabolism, shedding light on the mechanisms of vitamin B6 regulation in bacteria.
摘要：
在各种生物中，维生素B6，磷酸吡哆醛（PLP）的辅酶形式，由磷酸吡哆醇(PNP)合成。PNP水平的控制对于代谢稳态至关重要，因为PNP具有抑制PLP依赖性酶和蛋白质的潜力。尽管大肠杆菌中PNP代谢的唯一已知途径涉及PNP氧化酶的氧化，我们在大肠杆菌细胞裂解物中检测到强的PNP磷酸酶活性。为了鉴定未知的PNP磷酸酶，我们使用大肠杆菌serApdxH菌株进行了多拷贝抑制筛选，显示PNP依赖性条件杀伤力。结果表明，yigL基因的过表达，编码一种推定的糖磷酸酶，有效缓解了PNP的毒性。生化分析表明YigL对PNP具有很强的磷酸酶活性。YigL突变体表现出降低的PNP磷酸酶活性，细胞内PNP浓度升高，和增加的PNP敏感性，强调YigL在PNP稳态中的重要作用。YigL也显示与PLP的反应性。大肠杆菌细胞裂解物中PLP的磷酸酶活性被yigL的突变显著降低，而被ybhA的额外突变几乎消除，编码推定的PLP磷酸酶。这些结果强调了YigL的重要贡献，与YbhA结合，作为大肠杆菌中PNP和PLP去磷酸化的主要酶。IMPORTANCE磷酸吡哆醇（PNP）代谢对于维生素B6稳态和细胞代谢都至关重要。在大肠杆菌中，PNP的氧化是控制PNP水平的唯一已知机制。这项研究揭示了一种新的磷酸酶介导的PNP稳态机制。多种群抑制器筛选，酶的动力学分析，并且敲除/过表达研究将YigL鉴定为在大肠杆菌中面对升高的PNP浓度时有助于PNP体内平衡的关键PNP磷酸酶。这项研究还揭示了YigL的重大贡献，与YbhA结合，PLP代谢，阐明细菌中维生素B6的调节机制。
