Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 May;177(10):2804–2812. doi: 10.1128/jb.177.10.2804-2812.1995

Biochemical characterization of gapB-encoded erythrose 4-phosphate dehydrogenase of Escherichia coli K-12 and its possible role in pyridoxal 5'-phosphate biosynthesis.

G Zhao 1, A J Pease 1, N Bharani 1, M E Winkler 1
PMCID: PMC176952  PMID: 7751290

Abstract

One step in de novo pyridoxine (vitamin B6) and pyridoxal 5'-phosphate biosynthesis was predicted to be an oxidation catalyzed by an unidentified D-erythrose-4-phosphate dehydrogenase (E4PDH). To help identify this E4PDH, we purified the Escherichia coli K-12 gapA- and gapB-encoded dehydrogenases to homogeneity and tested whether either uses D-erythrose-4-phosphate (E4P) as a substrate. gapA (gap1) encodes the major D-glyceraldehyde-3-phosphate dehydrogenase (GA3PDH). The function of gapB (gap2) is unknown, although it was suggested that gapB encodes a second form of GA3PDH or is a cryptic gene. We found that the gapB-encoded enzyme is indeed an E4PDH and not a second GA3PDH, whereas gapA-encoded GA3PDH used E4P poorly, if at all, as a substrate under the in vitro reaction conditions used in this study. The amino terminus of purified E4PDH matched the sequence predicted from the gapB DNA sequence. Purified E4PDH was a heat-stable tetramer with a native molecular mass of 132 kDa. E4PDH had an apparent Km value for E4P [Kmapp(E4P)] of 0.96 mM, an apparent kcat catalytic constant for E4P [kcatapp(E4P)] of 200 s-1, Kmapp(NAD+) of 0.074 mM, and kcatapp(NAD+) of 169 s-1 in steady-state reactions in which NADH formation was determined. From specific activities in crude extracts, we estimated that there are at least 940 E4PDH tetramer molecules per bacterium growing in minimal salts medium plus glucose at 37 degrees C. Thin-layer chromatography confirmed that the product of the E4PDH reaction was likely the aldonic acid 4-phosphoerythronate. To establish a possible role of E4PDH in pyridoxal 5'-phosphate biosynthesis, we showed that 4-phosphoerythronate is a likely substrate for the 2-hydroxy-acid dehydrogenase encoded by the pdxB gene. Implications of these findings in the evolution of GA3PDHs are also discussed. On the basis of these results, we propose renaming gapB as epd (for D-erythrose-4-phosphate dehydrogenase).

Full Text

The Full Text of this article is available as a PDF (407.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alefounder P. R., Perham R. N. Identification, molecular cloning and sequence analysis of a gene cluster encoding the class II fructose 1,6-bisphosphate aldolase, 3-phosphoglycerate kinase and a putative second glyceraldehyde 3-phosphate dehydrogenase of Escherichia coli. Mol Microbiol. 1989 Jun;3(6):723–732. doi: 10.1111/j.1365-2958.1989.tb00221.x. [DOI] [PubMed] [Google Scholar]
  2. Arps P. J., Marvel C. C., Rubin B. C., Tolan D. A., Penhoet E. E., Winkler M. E. Structural features of the hisT operon of Escherichia coli K-12. Nucleic Acids Res. 1985 Jul 25;13(14):5297–5315. doi: 10.1093/nar/13.14.5297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blackmore P. F., Williams J. F., MacLeod J. K. Dimerization of erythrose 4-phosphate. FEBS Lett. 1976 Apr 15;64(1):222–226. doi: 10.1016/0014-5793(76)80288-8. [DOI] [PubMed] [Google Scholar]
  4. Bochner B. R., Ames B. N. Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography. J Biol Chem. 1982 Aug 25;257(16):9759–9769. [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Branlant G., Branlant C. Nucleotide sequence of the Escherichia coli gap gene. Different evolutionary behavior of the NAD+-binding domain and of the catalytic domain of D-glyceraldehyde-3-phosphate dehydrogenase. Eur J Biochem. 1985 Jul 1;150(1):61–66. doi: 10.1111/j.1432-1033.1985.tb08988.x. [DOI] [PubMed] [Google Scholar]
  7. Branlant G., Flesch G., Branlant C. Molecular cloning of the glyceraldehyde-3-phosphate dehydrogenase genes of Bacillus stearothermophilus and Escherichia coli, and their expression in Escherichia coli. Gene. 1983 Nov;25(1):1–7. doi: 10.1016/0378-1119(83)90161-0. [DOI] [PubMed] [Google Scholar]
  8. Camakaris J., Pittard J. Purification and properties of 3-deoxy-D-arabionheptulosonic acid-7-phosphate synthetase (trp) from Escherichia coli. J Bacteriol. 1974 Nov;120(2):590–597. doi: 10.1128/jb.120.2.590-597.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Conway T., Sewell G. W., Ingram L. O. Glyceraldehyde-3-phosphate dehydrogenase gene from Zymomonas mobilis: cloning, sequencing, and identification of promoter region. J Bacteriol. 1987 Dec;169(12):5653–5662. doi: 10.1128/jb.169.12.5653-5662.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. D'Alessio G., Josse J. Glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, and phosphoglyceromutase of Escherichia coli. Simultaneous purification and physical properties. J Biol Chem. 1971 Jul 10;246(13):4319–4325. [PubMed] [Google Scholar]
  11. Doolittle R. F., Feng D. F., Anderson K. L., Alberro M. R. A naturally occurring horizontal gene transfer from a eukaryote to a prokaryote. J Mol Evol. 1990 Nov;31(5):383–388. doi: 10.1007/BF02106053. [DOI] [PubMed] [Google Scholar]
  12. Drewke C., Notheis C., Hansen U., Leistner E., Hemscheidt T., Hill R. E., Spenser I. D. Growth response to 4-hydroxy-L-threonine of Escherichia coli mutants blocked in vitamin B6 biosynthesis. FEBS Lett. 1993 Mar 1;318(2):125–128. doi: 10.1016/0014-5793(93)80005-f. [DOI] [PubMed] [Google Scholar]
  13. Duncan K., Coggins J. R. The serC-aro A operon of Escherichia coli. A mixed function operon encoding enzymes from two different amino acid biosynthetic pathways. Biochem J. 1986 Feb 15;234(1):49–57. doi: 10.1042/bj2340049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Eikmanns B. J. Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomerase. J Bacteriol. 1992 Oct;174(19):6076–6086. doi: 10.1128/jb.174.19.6076-6086.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fraenkel D. G., Levisohn S. R. Glucose and gluconate metabolism in an Escherichia coli mutant lacking phosphoglucose isomerase. J Bacteriol. 1967 May;93(5):1571–1578. doi: 10.1128/jb.93.5.1571-1578.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. HANES C. S., ISHERWOOD F. A. Separation of the phosphoric esters on the filter paper chromatogram. Nature. 1949 Dec 31;164(4183):1107-12, illust. doi: 10.1038/1641107a0. [DOI] [PubMed] [Google Scholar]
  17. Hanahan D., Jessee J., Bloom F. R. Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol. 1991;204:63–113. doi: 10.1016/0076-6879(91)04006-a. [DOI] [PubMed] [Google Scholar]
  18. Heath J. D., Perkins J. D., Sharma B., Weinstock G. M. NotI genomic cleavage map of Escherichia coli K-12 strain MG1655. J Bacteriol. 1992 Jan;174(2):558–567. doi: 10.1128/jb.174.2.558-567.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hillman J. D., Fraenkel D. G. Glyceraldehyde 3-phosphate dehydrogenase mutants of Escherichia coli. J Bacteriol. 1975 Jun;122(3):1175–1179. doi: 10.1128/jb.122.3.1175-1179.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hughes K. T., Roth J. R. Conditionally transposition-defective derivative of Mu d1(Amp Lac). J Bacteriol. 1984 Jul;159(1):130–137. doi: 10.1128/jb.159.1.130-137.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. ISHII Y., HASHIMOTO T., TATIBANA M., YOSHIKAWA H. Erythronic acid 4-phosphate: an intermediate of inosine metabolism in human red cell hemolysates. Biochem Biophys Res Commun. 1963 Jan 18;10:19–22. doi: 10.1016/0006-291x(63)90260-2. [DOI] [PubMed] [Google Scholar]
  22. Irani M. H., Maitra P. K. Glyceraldehyde 3-p dehydrogenase, glycerate 3-P kinase and enolase mutants of Escherichia coli: genetic studies. Mol Gen Genet. 1976 Apr 23;145(1):65–71. doi: 10.1007/BF00331559. [DOI] [PubMed] [Google Scholar]
  23. Irani M., Maitra P. K. Isolation and characterization of Escherichia coli mutants defective in enzymes of glycolysis. Biochem Biophys Res Commun. 1974 Jan;56(1):127–133. doi: 10.1016/s0006-291x(74)80324-4. [DOI] [PubMed] [Google Scholar]
  24. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  25. Lam H. M., Tancula E., Dempsey W. B., Winkler M. E. Suppression of insertions in the complex pdxJ operon of Escherichia coli K-12 by lon and other mutations. J Bacteriol. 1992 Mar;174(5):1554–1567. doi: 10.1128/jb.174.5.1554-1567.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lam H. M., Winkler M. E. Characterization of the complex pdxH-tyrS operon of Escherichia coli K-12 and pleiotropic phenotypes caused by pdxH insertion mutations. J Bacteriol. 1992 Oct;174(19):6033–6045. doi: 10.1128/jb.174.19.6033-6045.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lam H. M., Winkler M. E. Metabolic relationships between pyridoxine (vitamin B6) and serine biosynthesis in Escherichia coli K-12. J Bacteriol. 1990 Nov;172(11):6518–6528. doi: 10.1128/jb.172.11.6518-6528.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Martin W., Brinkmann H., Savonna C., Cerff R. Evidence for a chimeric nature of nuclear genomes: eubacterial origin of eukaryotic glyceraldehyde-3-phosphate dehydrogenase genes. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8692–8696. doi: 10.1073/pnas.90.18.8692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McCandliss R. J., Poling M. D., Herrmann K. M. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. Purification and molecular characterization of the phenylalanine-sensitive isoenzyme from Escherichia coli. J Biol Chem. 1978 Jun 25;253(12):4259–4265. [PubMed] [Google Scholar]
  30. Nelson K., Whittam T. S., Selander R. K. Nucleotide polymorphism and evolution in the glyceraldehyde-3-phosphate dehydrogenase gene (gapA) in natural populations of Salmonella and Escherichia coli. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6667–6671. doi: 10.1073/pnas.88.15.6667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. PIZER L. I. THE PATHWAY AND CONTROL OF SERINE BIOSYNTHESIS IN ESCHERICHIA COLI. J Biol Chem. 1963 Dec;238:3934–3944. [PubMed] [Google Scholar]
  32. RACKER E., KLYBAS V., SCHRAMM M. Tetrose diphosphate, a specific inhibitor of glyceraldehyde 3-phosphate dehydrogenase. J Biol Chem. 1959 Oct;234:2510–2516. [PubMed] [Google Scholar]
  33. Roa B. B., Connolly D. M., Winkler M. E. Overlap between pdxA and ksgA in the complex pdxA-ksgA-apaG-apaH operon of Escherichia coli K-12. J Bacteriol. 1989 Sep;171(9):4767–4777. doi: 10.1128/jb.171.9.4767-4777.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schoenlein P. V., Roa B. B., Winkler M. E. Divergent transcription of pdxB and homology between the pdxB and serA gene products in Escherichia coli K-12. J Bacteriol. 1989 Nov;171(11):6084–6092. doi: 10.1128/jb.171.11.6084-6092.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schoner R., Herrmann K. M. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. Purification, properties, and kinetics of the tyrosine-sensitive isoenzyme from Escherichia coli. J Biol Chem. 1976 Sep 25;251(18):5440–5447. [PubMed] [Google Scholar]
  36. Soukri A., Mougin A., Corbier C., Wonacott A., Branlant C., Branlant G. Role of the histidine 176 residue in glyceraldehyde-3-phosphate dehydrogenase as probed by site-directed mutagenesis. Biochemistry. 1989 Mar 21;28(6):2586–2592. doi: 10.1021/bi00432a036. [DOI] [PubMed] [Google Scholar]
  37. Stephens C. M., Bauerle R. Analysis of the metal requirement of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli. J Biol Chem. 1991 Nov 5;266(31):20810–20817. [PubMed] [Google Scholar]
  38. Veiga-da-Cunha M., Santos H., Van Schaftingen E. Pathway and regulation of erythritol formation in Leuconostoc oenos. J Bacteriol. 1993 Jul;175(13):3941–3948. doi: 10.1128/jb.175.13.3941-3948.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. White R. S., Dempsey W. B. Purification and properties of vitamin B6 kinase from Escherichia coli B. Biochemistry. 1970 Oct 13;9(21):4057–4064. doi: 10.1021/bi00823a005. [DOI] [PubMed] [Google Scholar]
  40. Williams J. F., Blackmore P. F., Duke C. C., MacLeod J. K. Fact, uncertainty and speculation concerning the biochemistry of D-erythrose-4-phosphate and its metabolic roles. Int J Biochem. 1980;12(3):339–344. doi: 10.1016/0020-711x(80)90112-3. [DOI] [PubMed] [Google Scholar]
  41. Winicov I., Pizer L. I. The mechanism of end product inhibition of serine biosynthesis. IV. Subunit structure of phosphoglycerate dehydrogenase and steady state kinetic studies of phosphoglycerate oxidation. J Biol Chem. 1974 Mar 10;249(5):1348–1355. [PubMed] [Google Scholar]
  42. Woodruff W. W., 3rd, Wolfenden R. Inhibition of ribose-5-phosphate isomerase by 4-phosphoerythronate. J Biol Chem. 1979 Jul 10;254(13):5866–5867. [PubMed] [Google Scholar]
  43. Zhao G., Winkler M. E. An Escherichia coli K-12 tktA tktB mutant deficient in transketolase activity requires pyridoxine (vitamin B6) as well as the aromatic amino acids and vitamins for growth. J Bacteriol. 1994 Oct;176(19):6134–6138. doi: 10.1128/jb.176.19.6134-6138.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zhao G., Winkler M. E. Kinetic limitation and cellular amount of pyridoxine (pyridoxamine) 5'-phosphate oxidase of Escherichia coli K-12. J Bacteriol. 1995 Feb;177(4):883–891. doi: 10.1128/jb.177.4.883-891.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES