Overexpression, Crystallization, and Preliminary X-Ray Crystallographic Analysis of the Alanine Racemase from Enterococcus faecalis v583

  • Priyadarshi, Amit (Division of Biotechnology, College of Life Sciences & Biotechnology, Korea University) ;
  • Lee, Eun-Hye (Division of Biotechnology, College of Life Sciences & Biotechnology, Korea University) ;
  • Sung, Min-Woo (Division of Biotechnology, College of Life Sciences & Biotechnology, Korea University) ;
  • Kim, Jae-Hee (Division of Biotechnology, College of Life Sciences & Biotechnology, Korea University) ;
  • Ku, Min-Je (Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology) ;
  • Kim, Eunice Eun-Kyeong (Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology) ;
  • Hwang, Kwang-Yeon (Division of Biotechnology, College of Life Sciences & Biotechnology, Korea University)
  • Published : 2008.01.31

Abstract

Alanine racemase, a bacterial enzyme belonging to the fold-type III group of pyridoxal 5'-phosphate (PLP)-dependent enzymes, has been shown to catalyze the interconversion between L- and D-alanine. The alanine racemase from the pathogenic bacterium Enterococcus faecalis v583 has been overexpressed in E. coli and was shown to crystallize an enzyme at 295 K, using polyethylene glycol (PEG) 8000 as a precipitant. X-ray diffraction data to $2.5{\AA}$ has been collected using synchrotron radiation. The crystal is a member of the orthorhombic space group, $C222_1$ with unit cell parameter of a=94.634, b=156.516, $c=147.878{\AA},\;and\;{\alpha}={\beta}={\gamma}=90{\AA}$. Two or three monomers are likely to be present in the asymmetric unit, with a corresponding $V_m\; of\;3.38{\AA}^3\;Da^{-1}\;and\;2.26{\AA}^3\;Da^{-1}$ and a solvent content of 63.7% and 45.5%, respectively.

Keywords

References

  1. Bonten, M. J. M., C. A. Gaillard, F. H. V. Tiel, S. V. Geest, and E. E. Stobberingh. 1995. Colonization and infection with Enterococcus faecalis in intensive care units: The role of antimicrobial agents. Antimicrob. Agent. Chemother. 39: 2783-2786 https://doi.org/10.1128/AAC.39.12.2783
  2. Fenn, T. D., G. F. Stamper, A. A. Morollo, and D. Ringe. 2003. A side reaction of alanine racemase: Transamination of cycloserine. Biochemistry 42: 5775-5783 https://doi.org/10.1021/bi027022d
  3. 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
  4. Mustata, G. and J. M. Briggs. 2004. Cluster analysis of water molecules in alanine racemase and their putative structural role. Protein Eng. Des. Sel. 17: 223-234 https://doi.org/10.1093/protein/gzh033
  5. Mustata, G. I., T. A. Soares, and J. M. Briggs. 2003. Molecular dynamics studies of alanine racemase: A structural model for drug design. Biopolymers 70: 186-200 https://doi.org/10.1002/bip.10425
  6. Otwinowski, Z. and W. Minor. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276: 307-326 https://doi.org/10.1016/S0076-6879(97)76066-X
  7. Paulsen, I. T., L. Banerjei, G. S. A. Myers, K. E. Nelson, R. Seshadri, T. D. Read, D. E. Fouts, J. A. Eisen, S. R. Gill, J. F. Heidelberg, H. Tettelin, R. J. Dodson, L. Umayam, L. Brinkac, M. Beanan, S. Daugherty, R. T. DeBoy, S. Durkin, J. Kolonay, R. Madupu, W. Nelson, J. Vamathevan, B. Tran, J. Upton, T. Hansen, J. Shetty, H. Khouri, T. Utterback, D. Radune, K. A. Ketchum, B. A. Dougherty, and C. M. Fraser. 2003. Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis. Science 299: 2071-2074 https://doi.org/10.1126/science.1080613
  8. Shankar, N., C. V. Lockatell, A. S. Baghdayan, C. Drachenberg, M. S. Gilmore, and D. E. Johnson. 2001. Role of Enterococcus faecalis surface protein Esp in the pathogenesis of ascending urinary tract infection. Infect. Immune. 69: 4366-4372 https://doi.org/10.1128/IAI.69.7.4366-4372.2001
  9. Umeda, A., F. Garnier, P. Courvalin, and M. Galimand. 2002. Association between the vanB2 glycopeptide resistance operon and Tn1549 in enterococci from France. J. Antimicrob. Chemother. 50: 253-256 https://doi.org/10.1093/jac/dkf105
  10. Watanabe, A., T. Yoshimura, B. Mikami, H. Hayashi, H. Kagamiyama, and N. Esaki. 2002. Reaction mechanism of alanine racemase from Bacillus stearothermophilus: X-Ray crystallographic studies of the enzyme bound with N-(5'- phosphopyridoxyl)alanine. J. Biol. Chem. 277: 19166-19172 https://doi.org/10.1074/jbc.M201615200