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

다국어 초록 (Multilingual Abstract)

<B>ABSTRACT</B><P>d-Amino acid aminotransferases (d-AATs) from <I>Geobacillus toebii</I> SK1 and <I>Geobacillus</I> 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 <I>Bacilli</I> enzymes, with respective turnover rates of 47 and 55 s<SUP>−1</SUP> at 50°C. The <I>Geobacillus</I> enzymes were unique and shared limited sequence identities of below 45% with d-AATs from mesophilic and thermophilic <I>Bacillus</I> spp., except for a hypothetical protein with a 72% identity from the <I>G. kaustophilus</I> genome. Structural alignments showed that most key residues were conserved in the <I>Geobacillus</I> enzymes, although the conservative residues just before the catalytic lysine were distinctively changed: the 140-LRcD-143 sequence in <I>Bacillus</I>d-AATs was 144-EYcY-147 in the <I>Geobacillus</I>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 α-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 <I>Geobacillus</I>d-AATs had a π/π interaction that was replaceable with the salt bridge interaction between the arginine and aspartate residues in the LRcD sequence.</P>