1887

Abstract

Pyridoxal 5′-phosphate (PLP) is the active form of vitamin B and a cofactor for many essential metabolic processes such as amino acid biosynthesis and one carbon metabolism. 4’-deoxypyridoxine (4dPN) is a long known B antimetabolite but its mechanism of action was not totally clear. By exploring different conditions in which PLP metabolism is affected in the model organism K12, we showed that 4dPN cannot be used as a source of vitamin B as previously claimed and that it is toxic in several conditions where vitamin B homeostasis is affected, such as in a B auxotroph or in a mutant lacking the recently discovered PLP homeostasis gene, . In addition, we found that 4dPN sensitivity is likely the result of multiple modes of toxicity, including inhibition of PLP-dependent enzyme activity by 4’-deoxypyridoxine phosphate (4dPNP) and inhibition of cumulative pyridoxine (PN) uptake. These toxicities are largely dependent on the phosphorylation of 4dPN by pyridoxal kinase (PdxK).

Funding
This study was supported by the:
  • Sapenza University (Award RM12117A610B653E)
    • Principle Award Recipient: RobertoContestabile
  • National Institute of General Medical Sciences (Award GM129793)
    • Principle Award Recipient: Valériede Crécy-Lagard
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001319
2023-04-11
2024-05-21
Loading full text...

Full text loading...

/deliver/fulltext/micro/169/4/mic001319.html?itemId=/content/journal/micro/10.1099/mic.0.001319&mimeType=html&fmt=ahah

References

  1. Percudani R, Peracchi A. The B6 database: a tool for the description and classification of vitamin B6-dependent enzymatic activities and of the corresponding protein families. BMC Bioinformatics 2009; 10:273 [View Article] [PubMed]
    [Google Scholar]
  2. Breuer M, Earnest TM, Merryman C, Wise KS, Sun L et al. Essential metabolism for a minimal cell. Elife 2019; 8:1–75 [View Article] [PubMed]
    [Google Scholar]
  3. Lerma-Ortiz C, Jeffryes JG, Cooper AJL, Niehaus TD, Thamm AMK et al. “Nothing of chemistry disappears in biology”: the Top 30 damage-prone endogenous metabolites. Biochem Soc Trans 2016; 44:961–971 [View Article] [PubMed]
    [Google Scholar]
  4. Prunetti L, El Yacoubi B, Schiavon CR, Kirkpatrick E, Huang L et al. Evidence that COG0325 proteins are involved in PLP homeostasis. Microbiology 2016; 162:694–706 [View Article] [PubMed]
    [Google Scholar]
  5. Tramonti A, Nardella C, di Salvo M et al. Knowns and unknowns of vitamin B₆ metabolism in Escherichia coli. EcoSal Plus 2021; 9: [View Article]
    [Google Scholar]
  6. Lam HM, Tancula E, Dempsey WB, Winkler ME. Suppression of insertions in the complex pdxJ operon of Escherichia coli K-12 by lon and other mutations. J Bacteriol 1992; 174:1554–1567 [View Article] [PubMed]
    [Google Scholar]
  7. Yang Y, Tsui HCT, Man TK, Winkler ME. 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; 180:1814–1821 [View Article] [PubMed]
    [Google Scholar]
  8. Sévin DC, Fuhrer T, Zamboni N, Sauer U. Nontargeted in vitro metabolomics for high-throughput identification of novel enzymes in Escherichia coli. Nat Methods 2017; 14:187–194 [View Article] [PubMed]
    [Google Scholar]
  9. Ito T, Downs DM. Pyridoxal reductase, PdxI, is critical for salvage of pyridoxal in Escherichia coli. J Bacteriol 2020; 202:12 [View Article] [PubMed]
    [Google Scholar]
  10. 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 2018; 63:362–368 [View Article] [PubMed]
    [Google Scholar]
  11. Oya N. Pyridoxine uptake in Escherichia coli. Vitam 1970222–229
    [Google Scholar]
  12. Yamada R, Furukawa Y. Role of pyridoxal kinase in vitamin B₆ uptake by Escherichia coli. J Nutr Sci Vitaminol 1981; 27:177–191 [View Article] [PubMed]
    [Google Scholar]
  13. Yamada RH, Tsuji T, Nose Y. Uptake and utilization of vitamin B₆ and its phosphate esters by Escherichia coli. J Nutr Sci Vitaminol 1977; 23:7–17 [View Article] [PubMed]
    [Google Scholar]
  14. di Salvo ML, Safo MK, Contestabile R. Biomedical aspects of pyridoxal 5’-phosphate availability. Front Biosci 2012; 4:897–913 [View Article] [PubMed]
    [Google Scholar]
  15. di Salvo ML, Contestabile R, Safo MK. Vitamin B₆ salvage enzymes: mechanism, structure and regulation. Biochim Biophys Acta 2011; 1814:1597–1608 [View Article] [PubMed]
    [Google Scholar]
  16. Johnstone DL, Al-Shekaili HH, Tarailo-Graovac M, Wolf NI, Ivy AS et al. PLPHP deficiency: clinical, genetic, biochemical, and mechanistic insights. Brain 2019; 142:542–559 [View Article] [PubMed]
    [Google Scholar]
  17. Ito T, Iimori J, Takayama S, Moriyama A, Yamauchi A et al. Conserved pyridoxal protein that regulates Ile and Val metabolism. J Bacteriol 2013; 195:5439–5449 [View Article] [PubMed]
    [Google Scholar]
  18. Ito T, Hori R, Hemmi H, Downs DM, Yoshimura T. Inhibition of glycine cleavage system by pyridoxine 5’-phosphate causes synthetic lethality in glyA yggS and serA yggS in Escherichia coli. Mol Microbiol 2020; 113:270–284 [View Article] [PubMed]
    [Google Scholar]
  19. Scott TA, Hockney RC. Synthesis of vitamin B₆ by a mutant of Escherichia coli K12 and the action of 4’-deoxypyridoxine. J Gen Microbiol 1979; 110:285–289 [View Article] [PubMed]
    [Google Scholar]
  20. Sambrook J, Fritsch E, Maniatis T. Molecular Cloning: A Laboratory Manual. Plainview, NY: Cold Spring Harbor Laboratory; 1989 www.cshlpress.org accessed 18 August 2021
  21. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2006; 2:2006 [View Article] [PubMed]
    [Google Scholar]
  22. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci 2000; 97:6640–6645 [View Article] [PubMed]
    [Google Scholar]
  23. Moore SD. Assembling new Escherichia coli strains by transduction using phage P1. Methods Mol Biol 2011; 765:155–169 [View Article] [PubMed]
    [Google Scholar]
  24. Wetmore KM, Price MN, Waters RJ, Lamson JS, He J et al. Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons. mBio 2015; 6:e00306–15 [View Article] [PubMed]
    [Google Scholar]
  25. Price MN, Wetmore KM, Waters RJ, Callaghan M, Ray J et al. Mutant phenotypes for thousands of bacterial genes of unknown function. Nature 2018; 557:503–509 [View Article] [PubMed]
    [Google Scholar]
  26. Deatherage DE, Barrick JE. Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. Methods Mol Biol 2014; 1151:165–188 [View Article] [PubMed]
    [Google Scholar]
  27. Ghatge MS, Contestabile R, di Salvo ML, Desai JV, Gandhi AK et al. Pyridoxal 5’-phosphate is a slow tight binding inhibitor of E. coli pyridoxal kinase. PLoS One 2012; 7:e41680 [View Article] [PubMed]
    [Google Scholar]
  28. Contestabile R, Angelaccio S, Bossa F, Wright HT, Scarsdale N et al. Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase. Biochemistry 2000; 39:7492–7500 [View Article] [PubMed]
    [Google Scholar]
  29. Barile A, Tramonti A, di Salvo M et al. Allosteric feedback inhibition of pyridoxine 5′-phosphate oxidase from Escherichia coli. J Biol Chem 2019; 294:15593–15603 [View Article]
    [Google Scholar]
  30. Tramonti A, Nardella C, di Salvo ML, Barile A, Cutruzzolà F et al. Human cytosolic and mitochondrial serine hydroxymethyltransferase isoforms in comparison: full kinetic characterization and substrate inhibition properties. Biochemistry 2018; 57:6984–6996 [View Article] [PubMed]
    [Google Scholar]
  31. Coburn SP. The Chemistry and Metabolism of 4’ - Deoxypyridoxine Taylor & Francis Group; 2017 [View Article]
    [Google Scholar]
  32. Kim J, Flood JJ, Kristofich MR, Gidfar C, Morgenthaler AB et al. Hidden resources in the Escherichia coli genome restore PLP synthesis and robust growth after deletion of the essential gene pdxB. Proc Natl Acad Sci 2019; 116:24164–24173 [View Article] [PubMed]
    [Google Scholar]
  33. Lam HM, Winkler ME. Characterization of the complex pdxH-tyrS operon of Escherichia coli K-12 and pleiotropic phenotypes caused by pdxH insertion mutations. J Bacteriol 1992; 174:6033–6045 [View Article] [PubMed]
    [Google Scholar]
  34. Darin N, Reid E, Prunetti L, Samuelsson L, Husain RA et al. Mutations in PROSC disrupt cellular pyridoxal phosphate homeostasis and cause vitamin-B6-dependent epilepsy. Am J Hum Genet 2016; 99:1325–1337 [View Article] [PubMed]
    [Google Scholar]
  35. Bird TA, Levene CI. The effect of a vitamin B-6 antagonist, 4-deoxypyridoxine, on the cross-linking of collagen in the developing chick embryo. Biochem J 1983; 210:633–638 [View Article] [PubMed]
    [Google Scholar]
  36. Zhao G, Winkler ME. Kinetic limitation and cellular amount of pyridoxine (pyridoxamine) 5’-phosphate oxidase of Escherichia coli K-12. J Bacteriol 1995; 177:883–891 [View Article] [PubMed]
    [Google Scholar]
  37. Plamann MD, Stauffer GV. Characterization of the Escherichia coli gene for serine hydroxymethyltransferase. Gene 1983; 22:9–18 [View Article] [PubMed]
    [Google Scholar]
  38. Vu HN, Downs DM. Genetic analysis using vitamin B6 antagonist 4-deoxypyridoxine uncovers a connection between Pyridoxal 5’-phosphate and Coenzyme A metabolism in Salmonella enterica. J Bacteriol 2022; 204:e0060721 [View Article] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.001319
Loading
/content/journal/micro/10.1099/mic.0.001319
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Supplementary material 2

EXCEL

Supplementary material 3

EXCEL
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error