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Volume 146, Issue 7

Serine and alanine racemase activities of VanT: a protein necessary for vancomycin resistance in BM4174 Free

Abstract

Vancomycin resistance in results from the production of UDP-MurNAc-pentapeptide[D-Ser]. VanT, a membrane-bound serine racemase, is one of three proteins essential for this resistance. To investigate the selectivity of racemization of L-Ser or L-Ala by VanT, a strain of TKL-10 that requires D-Ala for growth at 42 °C was used as host for transformation experiments using plasmids containing the full-length from or the alanine racemase gene () of : both plasmids were able to complement TKL-10 at 42 °C. No alanine or serine racemase activities were detected in the host strain TKL-10 grown at 30, 34 or 37 °C. Serine and alanine racemase activities were found almost exclusively (96%) in the membrane fraction of TKL-10/pCA4(): the alanine racemase activity of VanT was 14% of the serine racemase activity in both TKL-10/pCA4() and XL-1 Blue/pCA4(). Alanine racemase activity was present mainly (95%) in the cytoplasmic fraction of TKL-10/pJW40(), with a trace (16%) of serine racemase activity. Additionally, DNA encoding the soluble domain of VanT was cloned and expressed in M15 as a His-tagged polypeptide and purified: this polypeptide also exhibited both serine and alanine racemase activities; the latter was approximately 18% of the serine racemase activity, similar to that of the full-length, membrane-bound enzyme. N-terminal sequencing of the purified His-tagged polypeptide revealed a single amino acid sequence, indicating that the formation of heterodimers between subunits of His-tagged C-VanT and endogenous alanine racemases from was unlikely. The authors conclude that the membrane-bound serine racemase VanT also has alanine racemase activity but is able to racemize serine more efficiently than alanine, and that the cytoplasmic domain is responsible for the racemase activity.

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Keyword(s): d-serine , Enterococcus gallinarum , racemases and vancomycin resistance
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2000-07-01
2024-04-25
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References

  1. Amann E., Brosius J. 1985; ‘ATG Vectors’ for regulated high-level expression of cloned genes in Escherichia coli. Gene 40:183–190 [CrossRef]
     [Google Scholar]
  2. Arias C. A., Martı́n-Martinez M., Blundell T. L., Arthur M., Courvalin P., Reynolds P. E. 1999; Characterization and modelling of VanT, a novel, membrane-bound, serine racemase from vancomycin-resistant Enterococcus gallinarum BM4174. Mol Microbiol 31:1653–1664 [CrossRef]
     [Google Scholar]
  3. Arias C. A., Courvalin P., Reynolds P. E. 2000; The vanC gene cluster of Enterococcus gallinarum BM4174. Antimicrob Agents Chemother 44:1660–1666 [CrossRef]
     [Google Scholar]
  4. Billot-Klein D., Gutmann L., Sable S., Guitet E., van Heijenoort J. 1994a; Modification of peptidoglycan precursors is a common feature of the low-level vancomycin-resistant VANB-type Enterococcus D366 and of the naturally glycopeptide-resistant species Lactobacillus casei, Pediococcus pentosaceus, Leuconostoc mesenteroides and Enterococcus gallinarum. J Bacteriol 176:2398–2405
     [Google Scholar]
  5. Billot-Klein D., Blanot D., Gutmann L., van Heijenoort J. 1994b; Association constants for the binding of vancomycin and teicoplanin to N-acetyl-d-alanyl-d-alanine and N-acetyl-d-alanyl-d-serine. Biochem J 304:1021–1022
     [Google Scholar]
  6. Blackwell J. R., Horgan R. 1991; A novel strategy for production of a highly expressed recombinant protein in an active form. FEBS Lett 295:10–12 [CrossRef]
     [Google Scholar]
  7. Blattner F. R., Burland V., Plunkett G., Sofia H. J., Daniels D. L. 1993; Analysis of the Escherichia coli genome. 4. DNA sequence of the region from 89·2 to 92·8 minutes. Nucleic Acids Res 21:5408–5417 [CrossRef]
     [Google Scholar]
  8. Bradford M. M. 1976; A rapid and sensitive method for rapid quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 72:248–254 [CrossRef]
     [Google Scholar]
  9. Bullock W. O., Fernandez J. M., Short J. M. 1987; XL-1 Blue: a high-efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. Biotechniques 5:376
     [Google Scholar]
  10. Corrigan J. J., Srinivasan N. G. 1966; The occurrence of certain d-amino acids in insects. Biochemistry 4:1185–1190
     [Google Scholar]
  11. Cosloy S. D., McFall E. 1973; Metabolism of d-serine in Escherichia coli K-12: mechanism of growth inhibition. J Bacteriol 114:685–694
     [Google Scholar]
  12. Dowhan W. Jr, Snell E. E. 1970; d-Serine dehydratase from Escherichia coli. II. Analytical studies and subunit structure. J Biol Chem 245:4618–4628
     [Google Scholar]
  13. Eidlic L., Neidhart F. C. 1965; Protein and nucleic acid synthesis in two mutants of Escherichia coli with temperature-sensitive aminoacyl ribonucleic acid synthetases. J Bacteriol 89:706–711
     [Google Scholar]
  14. Esaki N., Walsh C. T. 1986; Biosynthetic alanine racemase of Salmonella typhimurium: purification and characterization of the enzyme encoded by the alr gene. Biochemistry 25:3261–3267 [CrossRef]
     [Google Scholar]
  15. Friedman M. 1991; Formation, nutritional value, and safety of amino acids. In Nutritional and Toxicological Consequences of Food Processing pp. 447–481 New York: Plenum;
     [Google Scholar]
  16. Greene P. J., Maley F., Pedersen-Lane J., Santi D. V. 1993; Catalytically active cross-species heterodimers of thymidylate synthase. Biochemistry 32:10283–10288 [CrossRef]
     [Google Scholar]
  17. Gutnick D., Calvo J. M., Klopotowski T., Ames B. N. 1969; Compounds which serve as the sole source of carbon or nitrogen for Salmonella typhimurium LT-2. J Bacteriol 100:215–219
     [Google Scholar]
  18. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
     [Google Scholar]
  19. Lobocka M., Hennig J., Wild J., Klopotowski T. 1994; Organization and expression of the Escherichia coli K-12 dad operon encoding the smaller subunit of d-amino acid dehydrogenase and the catabolic alanine racemase. J Bacteriol 176:1500–1510
     [Google Scholar]
  20. McFall E., Newman E. B. 1996; Amino acids as carbon sources. In Escherichia coli and Salmonella: Cellular and Molecular Biology pp. 358–379Edited by Neidhart F. C.others Washington, DC: American Society for Microbiology;
     [Google Scholar]
  21. Messer J., Reynolds P. E. 1992; Modified peptidoglycan precursors produced by glycopeptide-resistant enterococci. FEMS Microbiol Lett 94:195–200 [CrossRef]
     [Google Scholar]
  22. Matsui T., Sekiguchi M., Hashimoto A., Tomita U., Nishikawa T., Wada K. 1995; Functional comparison of d-serine and glycine in rodents – the effect on cloned NMDA receptors and the extracellular concentration. J Neurochem 65:454–458
     [Google Scholar]
  23. Norrander J., Kempe T., Messing J. 1983; Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101–106 [CrossRef]
     [Google Scholar]
  24. Osterman A., Grishin N. V., Kinch L. N., Phillips M. A. 1994; Formation of functional cross-species heterodimers of ornithine decarboxylase. Biochemistry 33:13662–13667 [CrossRef]
     [Google Scholar]
  25. Park I. S., Chung-Hung L., Walsh C. T. 1997; Bacterial resistance to vancomycin: overproduction, purification and characterization of VanC-2 from Enterococcus casseliflavus as a d-Ala:d-Ser ligase. Proc Natl Acad Sci USA 94:10040–10044 [CrossRef]
     [Google Scholar]
  26. Reynolds P. E. 1989; Structure, biochemistry and mechanism of action of glycopeptide antibiotics. Eur J Clin Microbiol Infect Dis 8:943–950 [CrossRef]
     [Google Scholar]
  27. Reynolds P. E., Snaith H. A., Maguire A. J., Dutka-Malen S., Courvalin P. 1994; Analysis of peptidoglycan precursors in vancomycin-resistant Enterococcus gallinarum BM4174. Biochem J 301:5–8
     [Google Scholar]
  28. Reynolds P. E., Arias C. A., Courvalin P. 1999; Gene vanXY C encodes both dd-dipeptidase (VanX) and dd-carboxypeptidase (VanY) activities in vancomycin-resistant Enterococcus gallinarum BM4174. Mol Microbiol 36:341–349
     [Google Scholar]
  29. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  30. Shaw J. P., Petsko G. A., Ringe D. 1997; Determination of the structure of alanine racemase from Bacillus stearothermophilus at 1·9-Å resolution. Biochemistry 36:1329–1342 [CrossRef]
     [Google Scholar]
  31. Sun A. Q., Yuksel K. U., Gracet R. W. 1992; Interactions between the catalytic centers and subunit interface of triosephosphate isomerase probed by refolding, active-site modification, and subunit exchange. J Biol Chem 267:20168–20174
     [Google Scholar]
  32. Thompson J. D., Higgins D. G., Gibson T. J. 1994; clustalw: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680 [CrossRef]
     [Google Scholar]
  33. Truniger V., Boos W. 1994; Mapping and cloning of gldA, the structural gene of the Escherichia coli glycerol dehydrogenase. J Bacteriol 176:1796–1800
     [Google Scholar]
  34. Uo T., Yoshimura T., Shimizu S., Esaki N. 1998; Occurrence of pyridoxal 5′-phosphate-dependent serine racemase in silkworm, Bombyx mori. Biochem Biophys Res Commun 246:31–34 [CrossRef]
     [Google Scholar]
  35. Walsh C. T. 1989; Enzymes in the d-alanine branch of bacterial cell wall peptidoglycan assembly. J Biol Chem 264:2393–2396
     [Google Scholar]
  36. Wasserman S. A., Walsh C. T., Botstein D. 1983; Two alanine racemase genes in Salmonella typhimurium that differ in structure and function. J Bacteriol 153:1439–1450
     [Google Scholar]
  37. Wijsman H. J. W. 1972; The characterization of an alanine racemase mutant of Escherichia coli. Genet Res 20:269–277 [CrossRef]
     [Google Scholar]
  38. Wild J., Lobocka M., Walczak W., Klopotowski T. 1985; Identification of the dadX gene coding for the predominant isozyme of alanine racemase in Escherichia coli K-12. Mol Gen Genet 198:315–322 [CrossRef]
     [Google Scholar]
  39. Wolosker H., Shieth K. N., Takahashi M., Mothet J., Brady R. O. Jr, Ferris C. D., Snyder S. H. 1999a; Purification of serine racemase: biosynthesis of the neuromodulator d-serine. Proc Natl Acad Sci USA 96:721–725 [CrossRef]
     [Google Scholar]
  40. Wolosker H., Blackshaw S., Snyder S. H. 1999b; Serine racemase: a glial enzyme synthesizing d-serine to regulate glutamate-N-methyl-d-aspartate neurotransmission. Proc Natl Acad Sci USA 96:13409–13414 [CrossRef]
     [Google Scholar]
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Serine and alanine racemase activities of VanT: a protein necessary for vancomycin resistance in Enterococcus gallinarum BM4174
Microbiology 146, 1727 (2000); https://doi.org/10.1099/00221287-146-7-1727
/content/journal/micro/10.1099/00221287-146-7-1727
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