Identification of YigL As a PLP/PNP Phosphatase in

Overview

Journal Appl Environ Microbiol

Specialties Environmental Health
Microbiology

Date 2024 Aug 12

PMID 39133002

Authors

Hinano Matsuo

Naoki Yamada

Hisashi Hemmi

Tomokazu Ito

Affiliations

Soon will be listed here.

Abstract

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.

References

Yang Y, Tsui H, Man T, Winkler M . Identification and function of the pdxY gene, which encodes a novel pyridoxal kinase involved in the salvage pathway of pyridoxal 5'-phosphate biosynthesis in Escherichia coli K-12. J Bacteriol. 1998; 180(7):1814-21. PMC: 107095. DOI: 10.1128/JB.180.7.1814-1821.1998. View

Ito T . Role of the conserved pyridoxal 5'-phosphate-binding protein YggS/PLPBP in vitamin B6 and amino acid homeostasis. Biosci Biotechnol Biochem. 2022; 86(9):1183-1191. DOI: 10.1093/bbb/zbac113. View

Sugimoto R, Saito N, Shimada T, Tanaka K . Identification of YbhA as the pyridoxal 5'-phosphate (PLP) phosphatase in Escherichia coli: Importance of PLP homeostasis on the bacterial growth. J Gen Appl Microbiol. 2017; 63(6):362-368. DOI: 10.2323/jgam.2017.02.008. View

Ito T, Ogawa H, Hemmi H, Downs D, Yoshimura T . Mechanism of Pyridoxine 5'-Phosphate Accumulation in Pyridoxal 5'-Phosphate-Binding Protein Deficiency. J Bacteriol. 2022; 204(3):e0052121. PMC: 8923219. DOI: 10.1128/JB.00521-21. View

Pye V, Tingey A, Robson R, Moody P . The structure and mechanism of serine acetyltransferase from Escherichia coli. J Biol Chem. 2004; 279(39):40729-36. DOI: 10.1074/jbc.M403751200. View

Eliot A, Kirsch J . Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations. Annu Rev Biochem. 2004; 73:383-415. DOI: 10.1146/annurev.biochem.73.011303.074021. View

Mukherjee T, Hanes J, Tews I, Ealick S, Begley T . Pyridoxal phosphate: biosynthesis and catabolism. Biochim Biophys Acta. 2011; 1814(11):1585-96. DOI: 10.1016/j.bbapap.2011.06.018. View

Fonda M . Purification and characterization of vitamin B6-phosphate phosphatase from human erythrocytes. J Biol Chem. 1992; 267(22):15978-83. View

Vu H, Downs D . Loss of YggS (COG0325) impacts aspartate metabolism in Salmonella enterica. Mol Microbiol. 2021; 116(4):1232-1240. PMC: 8541914. DOI: 10.1111/mmi.14810. View

10.

Lee K, Jeong C, An Y, Lee H, Park S, Seok Y . FrsA functions as a cofactor-independent decarboxylase to control metabolic flux. Nat Chem Biol. 2011; 7(7):434-6. DOI: 10.1038/nchembio.589. View

11.

Prunetti L, El Yacoubi B, Schiavon C, Kirkpatrick E, Huang L, Bailly M . Evidence that COG0325 proteins are involved in PLP homeostasis. Microbiology (Reading). 2016; 162(4):694-706. DOI: 10.1099/mic.0.000255. View

12.

Sun Y, Vanderpool C . Physiological consequences of multiple-target regulation by the small RNA SgrS in Escherichia coli. J Bacteriol. 2013; 195(21):4804-15. PMC: 3807494. DOI: 10.1128/JB.00722-13. View

13.

Eberhardt J, Santos-Martins D, Tillack A, Forli S . AutoDock Vina 1.2.0: New Docking Methods, Expanded Force Field, and Python Bindings. J Chem Inf Model. 2021; 61(8):3891-3898. PMC: 10683950. DOI: 10.1021/acs.jcim.1c00203. View

14.

Ito T, Downs D . Pyridoxal Reductase, PdxI, Is Critical for Salvage of Pyridoxal in Escherichia coli. J Bacteriol. 2020; 202(12). PMC: 7253606. DOI: 10.1128/JB.00056-20. View

15.

Ito T, Yamamoto K, Hori R, Yamauchi A, Downs D, Hemmi H . Conserved Pyridoxal 5'-Phosphate-Binding Protein YggS Impacts Amino Acid Metabolism through Pyridoxine 5'-Phosphate in . Appl Environ Microbiol. 2019; 85(11). PMC: 6532037. DOI: 10.1128/AEM.00430-19. View

16.

Thomason L, Costantino N, Court D . E. coli genome manipulation by P1 transduction. Curr Protoc Mol Biol. 2008; Chapter 1:1.17.1-1.17.8. DOI: 10.1002/0471142727.mb0117s79. View

17.

Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M . Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2006; 2:2006.0008. PMC: 1681482. DOI: 10.1038/msb4100050. View

18.

Tobey K, Grant G . The nucleotide sequence of the serA gene of Escherichia coli and the amino acid sequence of the encoded protein, D-3-phosphoglycerate dehydrogenase. J Biol Chem. 1986; 261(26):12179-83. View

19.

Ito T, Iimori J, Takayama S, Moriyama A, Yamauchi A, Hemmi H . Conserved pyridoxal protein that regulates Ile and Val metabolism. J Bacteriol. 2013; 195(24):5439-49. PMC: 3889608. DOI: 10.1128/JB.00593-13. View

20.

Wilson M, Plecko B, Mills P, Clayton P . Disorders affecting vitamin B metabolism. J Inherit Metab Dis. 2019; 42(4):629-646. DOI: 10.1002/jimd.12060. View