1887

Abstract

The polymerization of lipid intermediate II by the transglycosylase activity of penicillin-binding proteins (PBPs) represents an important target for antibacterial action, but limited methods are available for quantitative assay of this reaction, or screening potential inhibitors. A new labelling method for lipid II polymerization products using Sanger’s reagent (fluoro-2,4-dinitrobenzene), followed by gel permeation HPLC analysis, has permitted the observation of intermediate polymerization products for monofunctional transglycosylase MGT. Peak formation is inhibited by 6 µM ramoplanin or enduracidin. Characterization by mass spectrometry indicates the formation of tetrasaccharide and octasaccharide intermediates, but not a hexasaccharide intermediate, suggesting a dimerization of a lipid-linked tetrasaccharide. Numerical modelling of the time-course data supports a kinetic model involving addition to lipid-linked tetrasaccharide of either lipid II or lipid-linked tetrasaccharide. Observation of free octasaccharide suggests that hydrolysis of the undecaprenyl diphosphate lipid carrier occurs at this stage in peptidoglycan transglycosylation.

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2014-08-01
2019-10-20
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References

  1. Barrett D., Wang T. S., Yuan Y., Zhang Y., Kahne D., Walker S.. ( 2007;). Analysis of glycan polymers produced by peptidoglycan glycosyltransferases. . J Biol Chem 282:, 31964–31971. [CrossRef][PubMed]
    [Google Scholar]
  2. Bertsche U., Breukink E., Kast T., Vollmer W.. ( 2005;). In vitro murein peptidoglycan synthesis by dimers of the bifunctional transglycosylase-transpeptidase PBP1B from Escherichia coli.. J Biol Chem 280:, 38096–38101. [CrossRef][PubMed]
    [Google Scholar]
  3. Bouhss A., Trunkfield A. E., Bugg T. D. H., Mengin-Lecreulx D.. ( 2008;). The biosynthesis of peptidoglycan lipid-linked intermediates. . FEMS Microbiol Rev 32:, 208–233. [CrossRef][PubMed]
    [Google Scholar]
  4. Breukink E., van Heusden H. E., Vollmerhaus P. J., Swiezewska E., Brunner L., Walker S., Heck A. J., de Kruijff B.. ( 2003;). Lipid II is an intrinsic component of the pore induced by nisin in bacterial membranes. . J Biol Chem 278:, 19898–19903. [CrossRef][PubMed]
    [Google Scholar]
  5. Bugg T. D. H., Braddick D., Dowson C. G., Roper D. I.. ( 2011;). Bacterial cell wall assembly: still an attractive antibacterial target. . Trends Biotechnol 29:, 167–173. [CrossRef][PubMed]
    [Google Scholar]
  6. Chen L., Walker D., Sun B., Hu Y., Walker S., Kahne D.. ( 2003;). Vancomycin analogues active against vanA-resistant strains inhibit bacterial transglycosylase without binding substrate. . Proc Natl Acad Sci U S A 100:, 5658–5663. [CrossRef][PubMed]
    [Google Scholar]
  7. Fang X., Tiyanont K., Zhang Y., Wanner J., Boger D., Walker S.. ( 2006;). The mechanism of action of ramoplanin and enduracidin. . Mol Biosyst 2:, 69–76. [CrossRef][PubMed]
    [Google Scholar]
  8. Heaslet H., Shaw B., Mistry A., Miller A. A.. ( 2009;). Characterization of the active site of S. aureus monofunctional glycosyltransferase (Mtg) by site-directed mutation and structural analysis of the protein complexed with moenomycin. . J Struct Biol 167:, 129–135. [CrossRef][PubMed]
    [Google Scholar]
  9. Hu Y., Helm J. S., Chen L., Ye X.-Y., Walker S.. ( 2003;). Ramoplanin inhibits bacterial transglycosylases by binding as a dimer to lipid II. . J Am Chem Soc 125:, 8736–8737. [CrossRef][PubMed]
    [Google Scholar]
  10. Huang C.-Y., Shih H.-W., Lin L.-Y., Tien Y.-W., Cheng T.-J. R., Cheng W.-C., Wong C.-H., Ma C.. ( 2012;). Crystal structure of Staphylococcus aureus transglycosylase in complex with a lipid II analog and elucidation of peptidoglycan synthesis mechanism. . Proc Natl Acad Sci U S A 109:, 6496–6501. [CrossRef][PubMed]
    [Google Scholar]
  11. Lee B.-S., Krisnanchettiar S., Lateef S. S., Lateef N. S., Gupta S.. ( 2005;). Oligosaccharide analyses of glycopeptides of horseradish peroxidase by thermal-assisted partial acid hydrolysis and mass spectrometry. . Carbohydr Res 340:, 1859–1865. [CrossRef][PubMed]
    [Google Scholar]
  12. Liu H., Wong C.-H.. ( 2006;). Characterization of a transglycosylase domain of Streptococcus pneumoniae PBP1b. . Bioorg Med Chem 14:, 7187–7195. [CrossRef][PubMed]
    [Google Scholar]
  13. Lloyd A. J., Gilbey A. M., Blewett A. M., De Pascale G., El Zoeiby A., Levesque R. C., Catherwood A. C., Tomasz A., Bugg T. D. H.. & other authors ( 2008;). Characterization of tRNA-dependent peptide bond formation by MurM in the synthesis of Streptococcus pneumoniae peptidoglycan. . J Biol Chem 283:, 6402–6417. [CrossRef][PubMed]
    [Google Scholar]
  14. Lovering A. L., de Castro L. H., Lim D., Strynadka N. C. J.. ( 2007;). Structural insight into the transglycosylation step of bacterial cell-wall biosynthesis. . Science 315:, 1402–1405. [CrossRef][PubMed]
    [Google Scholar]
  15. Lovering A. L., Safadi S. S., Strynadka N. C.. ( 2012;). Structural perspective of peptidoglycan biosynthesis and assembly. . Annu Rev Biochem 81:, 451–478. [CrossRef][PubMed]
    [Google Scholar]
  16. Offant J., Terrak M., Derouaux A., Breukink E., Nguyen-Distèche M., Zapun A., Vernet T.. ( 2010;). Optimization of conditions for the glycosyltransferase activity of penicillin-binding protein 1a from Thermotoga maritima.. FEBS J 277:, 4290–4298. [CrossRef]
    [Google Scholar]
  17. Reed P., Veiga H., Jorge A. M., Terrak M., Pinho M. G.. ( 2011;). Monofunctional transglycosylases are not essential for Staphylococcus aureus cell wall synthesis. . J Bacteriol 193:, 2549–2556. [CrossRef][PubMed]
    [Google Scholar]
  18. Sauvage E., Kerff F., Terrak M., Ayala J. A., Charlier P.. ( 2008;). The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis. . FEMS Microbiol Rev 32:, 234–258. [CrossRef][PubMed]
    [Google Scholar]
  19. Schouten J. A., Bagga S., Lloyd A. J., de Pascale G., Dowson C. G., Roper D. I., Bugg T. D. H.. ( 2006;). Fluorescent reagents for in vitro studies of lipid-linked steps of bacterial peptidoglycan biosynthesis: derivatives of UDPMurNAc-pentapeptide containing d-cysteine at position 4 or 5. . Mol Biosyst 2:, 484–491. [CrossRef][PubMed]
    [Google Scholar]
  20. Schwartz B., Markwalder J. A., Seitz S. P., Wang Y., Stein R. L.. ( 2002;). A kinetic characterization of the glycosyltransferase activity of Eschericia coli PBP1b and development of a continuous fluorescence assay. . Biochemistry 41:, 12552–12561. [CrossRef][PubMed]
    [Google Scholar]
  21. Sung M.-T., Lai Y.-T., Huang C.-Y., Chou L.-Y., Shih H.-W., Cheng W.-C., Wong C.-H., Ma C.. ( 2009;). Crystal structure of the membrane-bound bifunctional transglycosylase PBP1b from Escherichia coli.. Proc Natl Acad Sci U S A 106:, 8824–8829. [CrossRef][PubMed]
    [Google Scholar]
  22. Terrak M., Nguyen-Distèche M.. ( 2006;). Kinetic characterization of the monofunctional glycosyltransferase from Staphylococcus aureus. . J Bacteriol 188:, 2528–2532. [CrossRef][PubMed]
    [Google Scholar]
  23. Terrak M., Ghosh T. K., van Heijenoort J., Van Beeumen J., Lampilas M., Aszodi J., Ayala J. A., Ghuysen J.-M., Nguyen-Distèche M.. ( 1999;). The catalytic, glycosyl transferase and acyl transferase modules of the cell wall peptidoglycan-polymerizing penicillin-binding protein 1b of Escherichia coli.. Mol Microbiol 34:, 350–364. [CrossRef][PubMed]
    [Google Scholar]
  24. Vinatier V., Blakey C. B., Braddick D., Johnson B. R. G., Evans S. D., Bugg T. D. H.. ( 2009;). In vitro biosynthesis of bacterial peptidoglycan using d-Cys-containing precursors: fluorescent detection of transglycosylation and transpeptidation. . Chem Commun (Camb) 2009:, 4037–4039. [CrossRef][PubMed]
    [Google Scholar]
  25. Wang Q. M., Peery R. B., Johnson R. B., Alborn W. E., Yeh W.-K., Skatrud P. L.. ( 2001;). Identification and characterization of a monofunctional glycosyltransferase from Staphylococcus aureus.. J Bacteriol 183:, 4779–4785. [CrossRef][PubMed]
    [Google Scholar]
  26. Wang T. S., Manning S. A., Walker S., Kahne D.. ( 2008;). Isolated peptidoglycan glycosyltransferases from different organisms produce different glycan chain lengths. . J Am Chem Soc 130:, 14068–14069. [CrossRef][PubMed]
    [Google Scholar]
  27. Yuan Y., Barrett D., Zhang Y., Kahne D. E., Sliz P., Walker S.. ( 2007;). Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis. . Proc Natl Acad Sci U S A 104:, 5348–5353. [CrossRef][PubMed]
    [Google Scholar]
  28. Zhang Y., Fechter E. J., Wang T.-S. A., Barrett D., Walker S., Kahne D. E.. ( 2007;). Synthesis of heptaprenyl-lipid IV to analyze peptidoglycan glycosyltransferases. . J Am Chem Soc 129:, 3080–3081. [CrossRef][PubMed]
    [Google Scholar]
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