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Applied and Environmental Microbiology, February 2006, p. 1588-1594, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1588-1594.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Functional and Structural Characterization of Thermostable D-Amino Acid Aminotransferases from Geobacillus spp.

Seung-Goo Lee,^1, Seung-Pyo Hong,^2, Jae Jun Song,¹ Su-Jin Kim,¹ Mi-Sun Kwak,² and Moon-Hee Sung^2,3*

Laboratory of Microbial Function, KRIBB, Daejeon,¹ Bioleaders Corp., Daejeon,² Kookmin University, Seoul, Korea³

Received 5 July 2005/ Accepted 11 November 2005

D-Amino acid aminotransferases (D-AATs) from Geobacillus toebii SK1 and Geobacillus sp. strain KLS1 were cloned and characterized from a genetic, catalytic, and structural aspect. Although the enzymes were highly thermostable, their catalytic capability was approximately one-third of that of highly active Bacilli enzymes, with respective turnover rates of 47 and 55 s^–1 at 50°C. The Geobacillus enzymes were unique and shared limited sequence identities of below 45% with D-AATs from mesophilic and thermophilic Bacillus spp., except for a hypothetical protein with a 72% identity from the G. kaustophilus genome. Structural alignments showed that most key residues were conserved in the Geobacillus enzymes, although the conservative residues just before the catalytic lysine were distinctively changed: the 140-LRcD-143 sequence in Bacillus D-AATs was 144-EYcY-147 in the Geobacillus D-AATs. When the EYcY sequence from the SK1 enzyme was mutated into LRcD, a 68% increase in catalytic activity was observed, while the binding affinity toward -ketoglutarate decreased by half. The mutant was very close to the wild-type in thermal stability, indicating that the mutations did not disturb the overall structure of the enzyme. Homology modeling also suggested that the two tyrosine residues in the EYcY sequence from the Geobacillus D-AATs had a / interaction that was replaceable with the salt bridge interaction between the arginine and aspartate residues in the LRcD sequence.

Supplemental material for this article may be found at http://aem.asm.org/.

S.-G.L. and S.-P.H. contributed equally to this study.