Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
권호 표지

Crystallization and Preliminary X-Ray Crystallographic Analysis of UDP-N-Acetylglucosamine Enolpyruvyl Transferase from Haemophilus influenzae in Complex with UDP-N-Acetylglucosamine and Fosfomycin

  • Yoon, Hye-Jin (Department of Chemistry, College of Natural Sciences, Seoul National University) ;
  • Ku, Min-Je (Department of Chemistry, College of Natural Sciences, Seoul National University) ;
  • Ahn, Hyung Jun (Department of Chemistry, College of Natural Sciences, Seoul National University) ;
  • Lee, Byung Il (Department of Chemistry, College of Natural Sciences, Seoul National University) ;
  • Mikami, Bunzo (Laboratory of Quality Design and Exploitation, Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University) ;
  • Suh, Se Won (Department of Chemistry, College of Natural Sciences, Seoul National University)
  • Received : 2005.01.21
  • Accepted : 2005.02.23
  • Published : 2005.06.30
⟨ Previous Next ⟩

Abstract

The bacterial enzyme UDP-N-acetylglucosamine enolpyruvyl transferase catalyzes the first committed step of peptidoglycan biosynthesis, i.e., transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetyl-glucosamine. We have overexpressed the enzyme from Haemophilus influenzae in Escherichia coli and crystallized it in the apo-form, as well as in a complex with UDP-N-acetylglucosamine and fosfomycin using ammonium sulfate as the precipitant. X-ray diffraction data from a crystal of the apo-form were collected to $2.8{\AA}$ resolution at 293 K. The crystal quality was improved by co-crystallization with UDP-N-acetylglucosamine and fosfomycin. X-ray data to $2.2{\AA}$ have been collected at 100 K from a flash-frozen crystal of the complex. The complex crystals belong to the orthorhombic space group I222 (or $I2_12_12_1$) with unit-cell parameters of a = 63.7, b = 124.5, and $c=126.3{\AA}$. Assuming a monomer of the recombinant enzyme in the crystallographic asymmetric unit, the calculated Matthews parameter ($V_M$) is $2.71{\AA}^3Da^{-1}$ and solvent content is 54.6%.

Keywords

Acknowledgement

Supported by : Korea Ministry of Science and Technology, Korea Science and Engineering Foundation

References

  1. Baum, E. Z., Montenegro, D. A., Licata, L., Turchi, I., Webb, G. C., et al. (2001) Identification and characterization of new inhibitors of the Escherichia coli MurA enzyme. Antimicrob. Agents Chemother. 45, 3182-3188 https://doi.org/10.1128/AAC.45.11.3182-3188.2001
  2. Brown, E. D., Vivas, E. I., Walsh, C. T., and Kolter, R. (1995) MurA (MurZ), the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis, is essential in Escherichia coli. J. Bacteriol. 177, 4194-4197
  3. Bugg, T. D. and Walsh, C. T. (1992) Intracellular steps of bacterial cell wall peptidoglycan biosynthesis: enzymology, antibiotics, and antibiotic resistance. Nat. Prod. Rep. 9, 199-215 https://doi.org/10.1039/np9920900199
  4. Collaborative Computational Project, Number 4 (1994) The CCP4 suite: programs for protein crystallography. Acta Cryst. D50, 760-763
  5. Du, W., Brown, J. R., Sylvester, D. R., Huang, J., Chalker, A. F., et al. (2000) Two active forms of UDP-N-acetylglucosamine enolpyruvyl transferase in gram-positive bacteria. J. Bacteriol. 182, 4146-4152 https://doi.org/10.1128/JB.182.15.4146-4152.2000
  6. Eschenburg, S. and Schönbrunn, E. (2000) Comparative X-ray analysis of the un-liganded fosfomycin-target murA. Proteins 40, 290-298 https://doi.org/10.1002/(SICI)1097-0134(20000801)40:2<290::AID-PROT90>3.0.CO;2-0
  7. Fleischmann, R. D., Adams, M. D., White, O., Clayton, R. A., Kirkness, E. F., et al. (1995) Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269, 496-512 https://doi.org/10.1126/science.7542800
  8. Kahan, F. M., Kahan, J. S., Cassidy, P. J., and Kropp, H. (1974) The mechanism of action of fosfomycin (phosphonomycin). Ann. N. Y. Acad. Sci. 235, 364-386 https://doi.org/10.1111/j.1749-6632.1974.tb43277.x
  9. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 https://doi.org/10.1038/227680a0
  10. Leslie, A. G. W. (1994) MOSFLM User Guide, MRC-LMB, Cambridge, England
  11. Matthews, B. W. (1968) Solvent content of protein crystals. J. Mol. Biol. 33, 491-493 https://doi.org/10.1016/0022-2836(68)90205-2
  12. Schönbrunn, E., Sack, S., Eschenburg, S., Perrakis, A., Krekel, F., et al. (1996) Crystal structure of UDP-N-acetylglucosamine enolpyruvyltransferase, the target of the antibiotic fosfomycin. Structure 4, 1065-1075 https://doi.org/10.1016/S0969-2126(96)00113-X
  13. Skarzynski, T., Mistry, A., Wonacott, A., Hutchinson, S. E., Kelly, V. A., et al. (1996) Structure of UDP-N-acetylglucosamine enolpyruvyl transferase, an enzyme essential for the synthesis of bacterial peptidoglycan, complexed with substrate UDP-N-acetylglucosamine and the drug fosfomycin. Structure 4, 1465-1474 https://doi.org/10.1016/S0969-2126(96)00153-0