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

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, aspa rtate, and glutamine. The cloned gene was expressed and the protein was pur ified to homogeneity. The A. pyrophilus racemase is present as a dimer but it oligomerizes as the concentration of salt is increased. The K-m and k(ca t) values of the overexpressed A. pyrophilus glutamate racemase for the rac emization 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 o f the racemase activity by treatment with cysteine-modifying reagents sugge sts that cysteine residues may be important for activity. The protein shows strong thermostability in the presence of phosphate ion, and it retains mo re than 50% of its activity after incubation at 85 degrees C for 90 min.