Identification of YigL as a PLP/PNP phosphatase in <i>Escherichia coli</i>

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 <i>Escherichia coli</i> involves oxidation by PNP oxidase, we detected a strong PNP phosphatase activity in <i>E. coli</i> cell lysate. To identify the unknown PNP phosphatase(s), we performed a multicopy suppressor screening using the <i>E. coli serA pdxH</i> strain, which displays PNP-dependent conditional lethality. The results showed that overexpression of the <i>yigL</i> gene, encoding a putative sugar phosphatase, effectively alleviated the PNP toxicity. Biochemical analysis revealed that YigL has strong phosphatase activity against PNP. A <i>yigL</i> 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 <i>E. coli</i> cell lysate was significantly reduced by mutation of <i>yigL</i> and nearly abolished by additional mutation of <i>ybhA</i>, 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 <i>E. coli</i>.IMPORTANCEPyridoxine phosphate (PNP) metabolism is critical for both vitamin B₆ homeostasis and cellular metabolism. In <i>Escherichia coli</i>, 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 <i>E. coli</i>. 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.