Two Amino Acid Racemase from Aquifex pyrophilus, a Hyperthermophilic Bacteria
Sang-Suk KIMa, In-Geol CHOIa, Sung-Hou KIMb and Yeon G. YUb
a Structural Biology Center, Korea Institute of Science and Technology, 39-1 Hwawolkok- Dong, Songbuk-Ku, Seoul Korea
b Department of Chemistry and Lawrence Berkeley National Laboratory, University of California, Berkeley, California, CA94720, USA
Two genes encoding glutamate racemase and alanine racemase are cloned from Aquifex pyrophilus, a hyperthermophilic bacteria. The deduced amino acid sequence from the open reading frame of the genes shows strong sequence similarities to each corresponding racemases. The proteins of the cloned genes have been expressed in Escherichia coli, purified in homogeneity and examined their activity. Glutamate racemase presents as monomeric protein of 28 kDa and uses only glutamate as a substrate and converts L-glutamate to D-glutamate or vice versa. However it has 80 times higher specific activity for L-glutamate than for D-glutamate. The protein has two cystein at the highly conserved regions and its racemase activity is sensitive to cystein modifying reagents indicating that cystein residues are important for the activity, probably serve as bases for deprotonation reaction. Alanine racemase homologue also show racemase activity against alanine. Both racemases show strong heat resistance like other proteins from hyperthermophiles. Aquifex glutamate racemase has an optimum temperature for activity of 80℃ and maintains more than 90% of its activity after 24 hr incubation at 60℃ or 65 % after 3 hr incubation at 80℃.
