Department of Applied Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furou-chou, Chikusa, Nagoya, Aichi, Japan.
Appl Environ Microbiol. 2024 Sep 18;90(9):e0127024. doi: 10.1128/aem.01270-24. Epub 2024 Aug 12.
In various organisms, the coenzyme form of vitamin B, 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 involves oxidation by PNP oxidase, we detected a strong PNP phosphatase activity in cell lysate. To identify the unknown PNP phosphatase(s), we performed a multicopy suppressor screening using the strain, which displays PNP-dependent conditional lethality. The results showed that overexpression of the gene, encoding a putative sugar phosphatase, effectively alleviated the PNP toxicity. Biochemical analysis revealed that YigL has strong phosphatase activity against PNP. A 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 cell lysate was significantly reduced by mutation of and nearly abolished by additional mutation of , 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 .IMPORTANCEPyridoxine phosphate (PNP) metabolism is critical for both vitamin B homeostasis and cellular metabolism. In , 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 . This study also revealed a significant contribution of YigL, in combination with YbhA, to PLP metabolism, shedding light on the mechanisms of vitamin B regulation in bacteria.
在各种生物体中，辅酶形式的维生素 B，即吡哆醛磷酸（PLP），是由吡哆醇磷酸（PNP）合成的。PNP 水平的控制对于代谢稳态至关重要，因为 PNP 有可能抑制依赖 PLP 的酶和蛋白质。尽管目前已知在 中 PNP 代谢的唯一途径是由 PNP 氧化酶氧化，但我们在 细胞裂解物中检测到一种强烈的 PNP 磷酸酶活性。为了鉴定未知的 PNP 磷酸酶，我们使用了显示 PNP 依赖性条件致死性的 菌株进行了多拷贝抑制子筛选。结果表明，编码一种假定的糖磷酸酶的 基因的过表达有效地缓解了 PNP 的毒性。生化分析表明，YigL 对 PNP 具有很强的磷酸酶活性。 突变体表现出降低的 PNP 磷酸酶活性、升高的细胞内 PNP 浓度和增加的 PNP 敏感性，突出了 YigL 在 PNP 稳态中的重要作用。YigL 还与 PLP 反应。 细胞裂解物中 PLP 的磷酸酶活性因 突变而显著降低，因额外突变编码假定的 PLP 磷酸酶的 而几乎被废除。这些结果强调了 YigL 与 YbhA 相结合作为 中 PNP 和 PLP 去磷酸化的主要酶的重要贡献。
