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. 1996 Jan;178(1):232–239. doi: 10.1128/jb.178.1.232-239.1996

A novel alpha-ketoglutarate reductase activity of the serA-encoded 3-phosphoglycerate dehydrogenase of Escherichia coli K-12 and its possible implications for human 2-hydroxyglutaric aciduria.

G Zhao 1, M E Winkler 1
PMCID: PMC177644  PMID: 8550422

Abstract

Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. The SerA enzyme is evolutionarily related to the pdxB gene product, 4-phosphoerythronate dehydrogenase, which catalyzes the second step in one branch of pyridoxal 5'-phosphate coenzyme biosynthesis. Both the Ser and pyridoxal 5'-phosphate biosynthetic pathways use the serC(pdxF)-encoded transaminase in their next steps. In an analysis of these parallel pathways, we attempted to couple the transaminase and dehydrogenase reactions in the reverse direction. Unexpectedly, we found that the SerA enzyme catalyzes a previously undetected reduction of alpha-ketoglutarate (alpha KG) to 2-hydroxyglutaric acid (HGA). Numerous criteria ruled out the possibility that this SerA alpha KG reductase activity was caused by contamination in the substrate or purified enzyme preparations. HGA was confirmed as the product of the SerA alpha KG reductase reaction by thin-layer chromatography and by enzyme assays showing that both the D- and L-isomers of HGA were substrates for the reverse (dehydrogenase) reaction. Detailed steady-state kinetic analyses showed that alpha KG reduction (apparent Michaelis-Menten constant [Km(app)] = 88 microM; apparent catalytic constant [kcat(app)] = 33.3 s-1) and 3-phosphohydroxypyruvate reduction (Km(app) = 3.2 microM; kcatapp = 27.8 s-1), which is the reverse reaction of 3PG oxidation, were the major in vitro activities of the SerA enzyme. The SerA alpha KG reductase was inhibited by Ser, D-HGA, 3PG, and glycine (Gly), whereas the D-HGA dehydrogenase was inhibited by Ser, alpha KG, 3-phosphohydroxypyruvate, and Gly. The implications of these findings for the regulation of Ser biosynthesis, the recycling of NADH, and the enzymology of 2-hydroxyacid dehydrogenases are discussed. Since the same pathway of Ser biosynthesis seems to be present in all organisms, these results suggest that a mutation in the human SerA homolog may contribute to the neurometabolic diseases D- and L-2-hydroxyglutaric aciduria, which lead to the accumulation of D-HGA and L-HGA, respectively.

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Selected References

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  1. Barth P. G., Hoffmann G. F., Jaeken J., Lehnert W., Hanefeld F., van Gennip A. H., Duran M., Valk J., Schutgens R. B., Trefz F. K. L-2-hydroxyglutaric acidemia: a novel inherited neurometabolic disease. Ann Neurol. 1992 Jul;32(1):66–71. doi: 10.1002/ana.410320111. [DOI] [PubMed] [Google Scholar]
  2. Barth P. G., Hoffmann G. F., Jaeken J., Wanders R. J., Duran M., Jansen G. A., Jakobs C., Lehnert W., Hanefeld F., Valk J. L-2-hydroxyglutaric acidaemia: clinical and biochemical findings in 12 patients and preliminary report on L-2-hydroxyacid dehydrogenase. J Inherit Metab Dis. 1993;16(4):753–761. doi: 10.1007/BF00711907. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Bridgers W. F. The biosynthesis of serine in mouse brain extracts. J Biol Chem. 1965 Dec;240(12):4591–4597. [PubMed] [Google Scholar]
  6. Chalmers R. A., Lawson A. M., Watts R. W., Tavill A. S., Kamerling J. P., Hey E., Ogilvie D. D-2-hydroxyglutaric aciduria: case report and biochemical studies. J Inherit Metab Dis. 1980;3(1):11–15. doi: 10.1007/BF02312516. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Duran M., Kamerling J. P., Bakker H. D., van Gennip A. H., Wadman S. K. L-2-Hydroxyglutaric aciduria: an inborn error of metabolism? J Inherit Metab Dis. 1980;3(4):109–112. doi: 10.1007/BF02312543. [DOI] [PubMed] [Google Scholar]
  9. Gibson K. M., Craigen W., Herman G. E., Jakobs C. D-2-hydroxyglutaric aciduria in a newborn with neurological abnormalities: a new neurometabolic disorder? J Inherit Metab Dis. 1993;16(3):497–500. doi: 10.1007/BF00711664. [DOI] [PubMed] [Google Scholar]
  10. Gibson K. M., ten Brink H. J., Schor D. S., Kok R. M., Bootsma A. H., Hoffmann G. F., Jakobs C. Stable-isotope dilution analysis of D- and L-2-hydroxyglutaric acid: application to the detection and prenatal diagnosis of D- and L-2-hydroxyglutaric acidemias. Pediatr Res. 1993 Sep;34(3):277–280. doi: 10.1203/00006450-199309000-00007. [DOI] [PubMed] [Google Scholar]
  11. Grant G. A. A new family of 2-hydroxyacid dehydrogenases. Biochem Biophys Res Commun. 1989 Dec 29;165(3):1371–1374. doi: 10.1016/0006-291x(89)92755-1. [DOI] [PubMed] [Google Scholar]
  12. Grant G. A., Bradshaw R. A. D-3-Phosphoglycerate dehydrogenase from chicken liver. II. Chemical and physical properties. J Biol Chem. 1978 Apr 25;253(8):2727–2731. [PubMed] [Google Scholar]
  13. Grant G. A., Keefer L. M., Bradshaw R. A. D-3-Phosphoglycerate dehydrogenase from chicken liver. I. Purification. J Biol Chem. 1978 Apr 25;253(8):2724–2726. [PubMed] [Google Scholar]
  14. Itoh H., Dempsey W. B. Purification of 3-phosphoserine-alpha-ketoglutarate transaminase from Escherichia coli B. Life Sci II. 1970 Nov 22;9(22):1289–1294. doi: 10.1016/0024-3205(70)90128-1. [DOI] [PubMed] [Google Scholar]
  15. Kaufman E. E., Nelson T., Fales H. M., Levin D. M. Isolation and characterization of a hydroxyacid-oxoacid transhydrogenase from rat kidney mitochondria. J Biol Chem. 1988 Nov 15;263(32):16872–16879. [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Lowry O. H., Carter J., Ward J. B., Glaser L. The effect of carbon and nitrogen sources on the level of metabolic intermediates in Escherichia coli. J Biol Chem. 1971 Nov;246(21):6511–6521. [PubMed] [Google Scholar]
  19. McKitrick J. C., Pizer L. I. Regulation of phosphoglycerate dehydrogenase levels and effect on serine synthesis in Escherichia coli K-12. J Bacteriol. 1980 Jan;141(1):235–245. doi: 10.1128/jb.141.1.235-245.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Moses V., Sharp P. B. Intermediary metabolite levels in Escherichia coli. J Gen Microbiol. 1972 Jun;71(1):181–190. doi: 10.1099/00221287-71-1-181. [DOI] [PubMed] [Google Scholar]
  21. PIZER L. I. ENZYMOLOGY AND REGULATION OF SERINE BIOSYNTHESIS IN CULTURED HUMAN CELLS. J Biol Chem. 1964 Dec;239:4219–4226. [PubMed] [Google Scholar]
  22. PIZER L. I., POTOCHNY M. L. NUTRITIONAL AND REGULATORY ASPECTS OF SERINE METABOLISM IN ESCHERICHIA COLI. J Bacteriol. 1964 Sep;88:611–619. doi: 10.1128/jb.88.3.611-619.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. PIZER L. I. THE PATHWAY AND CONTROL OF SERINE BIOSYNTHESIS IN ESCHERICHIA COLI. J Biol Chem. 1963 Dec;238:3934–3944. [PubMed] [Google Scholar]
  24. Pizer L. I. Comparative enzymology of serine biosynthesis in mammalian systems. Biochim Biophys Acta. 1966 Aug 24;124(2):418–420. doi: 10.1016/0304-4165(66)90212-1. [DOI] [PubMed] [Google Scholar]
  25. Pizer L. I., Regan J. D. Basis for the serine requirement in leukemic and normal human leukocytes. Reduced levels of the enzymes in the phosphorylated pathway. J Natl Cancer Inst. 1972 Jun;48(6):1897–1900. [PubMed] [Google Scholar]
  26. REEVES H. C., AJL S. J. Alpha-hydroxyglutaric acid synthetase. J Bacteriol. 1962 Jul;84:186–187. doi: 10.1128/jb.84.1.186-187.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Schuller D. J., Fetter C. H., Banaszak L. J., Grant G. A. Enhanced expression of the Escherichia coli serA gene in a plasmid vector. Purification, crystallization, and preliminary X-ray data of D-3 phosphoglycerate dehydrogenase. J Biol Chem. 1989 Feb 15;264(5):2645–2648. [PubMed] [Google Scholar]
  29. Schuller D. J., Grant G. A., Banaszak L. J. The allosteric ligand site in the Vmax-type cooperative enzyme phosphoglycerate dehydrogenase. Nat Struct Biol. 1995 Jan;2(1):69–76. doi: 10.1038/nsb0195-69. [DOI] [PubMed] [Google Scholar]
  30. Siegel W. H., Donohue T., Bernlohr R. W. Determination of pools of tricarboxylic acid cycle and related acids in bacteria. Appl Environ Microbiol. 1977 Nov;34(5):512–517. doi: 10.1128/aem.34.5.512-517.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Slaughter J. C., Davies D. D. The isolation and characterization of 3-phosphoglycerate dehydrogenase from peas. Biochem J. 1968 Oct;109(5):743–748. doi: 10.1042/bj1090743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sugimoto E., Pizer L. I. The mechanism of end product inhibition of serine biosynthesis. I. Purification and kinetics of phosphoglycerate dehydrogenase. J Biol Chem. 1968 May 10;243(9):2081–2089. [PubMed] [Google Scholar]
  33. Sugimoto E., Pizer L. I. The mechanism of end product inhibition of serine biosynthesis. II. Optical studies of phosphoglycerate dehydrogenase. J Biol Chem. 1968 May 10;243(9):2090–2098. [PubMed] [Google Scholar]
  34. Taguchi H., Ohta T. D-lactate dehydrogenase is a member of the D-isomer-specific 2-hydroxyacid dehydrogenase family. Cloning, sequencing, and expression in Escherichia coli of the D-lactate dehydrogenase gene of Lactobacillus plantarum. J Biol Chem. 1991 Jul 5;266(19):12588–12594. [PubMed] [Google Scholar]
  35. Vinals C., Depiereux E., Feytmans E. Prediction of structurally conserved regions of D-specific hydroxy acid dehydrogenases by multiple alignment with formate dehydrogenase. Biochem Biophys Res Commun. 1993 Apr 15;192(1):182–188. doi: 10.1006/bbrc.1993.1398. [DOI] [PubMed] [Google Scholar]
  36. Walsh D. A., Sallach H. J. D-3-phosphoglycerate dehydrogenase. Further studies on the enzyme isolated from chicken liver. Biochim Biophys Acta. 1967 Sep 12;146(1):26–34. doi: 10.1016/0005-2744(67)90070-8. [DOI] [PubMed] [Google Scholar]
  37. Walsh D. A., Sallach H. J. Purification and properties of chicken liver D-3-phosphoglycerate dehydrogenase. Biochemistry. 1965 Jun;4(6):1076–1085. doi: 10.1021/bi00882a015. [DOI] [PubMed] [Google Scholar]
  38. Winicov I. Stereospecificity of hydrogen transfer by phosphoglycerate dehydrogenase. Biochim Biophys Acta. 1975 Aug 26;397(2):288–293. doi: 10.1016/0005-2744(75)90118-7. [DOI] [PubMed] [Google Scholar]
  39. Zhao G., Pease A. J., Bharani N., Winkler M. E. Biochemical characterization of gapB-encoded erythrose 4-phosphate dehydrogenase of Escherichia coli K-12 and its possible role in pyridoxal 5'-phosphate biosynthesis. J Bacteriol. 1995 May;177(10):2804–2812. doi: 10.1128/jb.177.10.2804-2812.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]

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