Extremophiles

, Volume 3, Issue 3, pp 175–183

Molecular cloning, expression, and characterization of a thermostable glutamate racemase from a hyperthermophilic bacterium, Aquifex pyrophilus

  • Sang Suk Kim
  • I.-G. Choi
  • Sung-Hou Kim
  • Y. G. Yu
ORIGINAL PAPER

DOI: 10.1007/s007920050114

Cite this article as:
Kim, S., Choi, I., Kim, S. et al. Extremophiles (1999) 3: 175. doi:10.1007/s007920050114

Abstract

A gene encoding glutamate racemase has been cloned from Aquifex pyrophilus, a hyperthermophilic bacterium, and expressed in Escherichia coli. The A. pyrophilus glutamate racemase is composed of 254 amino acids and shows high homology with glutamate racemase from Escherichia coli, Bacillus subtilis, or Lactobacillus brevis. This racemase converts l- or d-glutamate to d- or l-glutamate, respectively, but not other amino acids such as alanine, aspartate, and glutamine. The cloned gene was expressed and the protein was purified to homogeneity. The A. pyrophilus racemase is present as a dimer but it oligomerizes as the concentration of salt is increased. The Km and kcat values of the overexpressed A. pyrophilus glutamate racemase for the racemization of l-glutamate to the d-form and the conversion of d-glutamate to the l-form were measured as 1.8 ± 0.4 mM and 0.79 ± 0.06 s−1 or 0.50 ± 0.07 mM and 0.25 ± 0.01 s−1, respectively. Complete inactivation of the racemase activity by treatment with cysteine-modifying reagents suggests that cysteine residues may be important for activity. The protein shows strong thermostability in the presence of phosphate ion, and it retains more than 50% of its activity after incubation at 85°C for 90 min.

Key wordsAquifex pyrophilusMolecular cloningGlutamate racemaseOverexpressionThermostability

Copyright information

© Springer-Verlag Tokyo 1999

Authors and Affiliations

  • Sang Suk Kim
    • 1
  • I.-G. Choi
    • 1
  • Sung-Hou Kim
    • 1
  • Y. G. Yu
    • 1
  1. 1.Structural Biology Center, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul, Korea Tel. +82-2-958-5936; Fax +82-2-958-5939 e-mail: ygy@kistmail.kist.re.krKR
  2. 2.Department of Chemistry and Lawrence Berkeley National Laboratory, University of California, Berkeley, CA, USAUS