Biochemical and structural characterization of a highly active branched-chain amino acid aminotransferase from Pseudomonas sp. for efficient biosynthesis of chiral amino acids

Aminotransferases (ATs) are important biocatalysts for the synthesis of chiral amines because of their capability of introducing amino group into ketones or keto acids as well as their high enantioselectivity, high regioselectivity and no requirement of external addition of cofactor. Among all ATs, branched-chain amino acid aminotransferase (BCAT) can reversibly catalyse branched-chain amino acids (BCAAs), including L -valine, L -leucine, and L -isoleucine, with α-ketoglutaric acid to form the corresponding ketonic acids and L -glutamic acid. Alternatively, BCATs have been used for the biosynthesis of unnatural amino acids, such as L -tert-leucine. In the present study, the BCAT from Pseudomonas sp. (PsBCAT) was cloned and expressed in Escherichia coli for biochemical and structural analyses. The optimal reaction temperature and pH of PsBCAT were 40 °C and 8.5, respectively. PsBCAT exhibited a comparatively broader substrate spectrum, and showed remarkably high activity with L -leucine, L -valine, L -isoleucine and L -methionine with activities of 105 U/mg, 127 U/mg, 115 U/mg and 98 U/mg, respectively. Additionally, PsBCAT had activities with aromatic L -amino acids, L -histidine, L -lysine, and L -threonine. To analyse the catalytic mechanism of PsBCAT with the broad substrate spectrum, the crystal structure of PsBCAT was also determined. Finally, conjugated with the ornithine aminotransferase (OrnAT) from Bacillus subtilis, the coupled system was applied to the preparation of L -tert-leucine with 83% conversion, which provided an approximately 2.7-fold higher yield than the single BCAT reaction.