Investigation of the metabolic role of d-amino acid aminotransferase in Arabidopsis thaliana using a column-switching two-dimensional high-performance liquid chromatography system

To investigate the physiological role of Arabidopsis thaliana D-amino acid aminotransferase (AtDAAT), which catalyzes the transformation of D-aspartate (D-Asp) to D-glutamate (D-Glu) and D-alanine (D-Ala), we analyzed the amounts of various D-amino acids in AtDAAT-deficient and wild-type A. thaliana grown in a medium containing D-Asp. The amounts of Asp, Glu, Ala, valine (Val), and leucine (Leu) enantiomers in AtDAAT-deficient and wild-type A. thaliana were determined using a highly selective two-dimensional high-performance liquid chromatography (2D-HPLC) system. The 2D-HPLC system comprised reversed-phase and chiral separation columns, and amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole for detection. To analyze the various d- and l-amino acids in A. thaliana, the separation conditions reported in our previous study were applied with modifications, and the method was validated. Enantioseparation, linearity, and accuracy were satisfactory for all the amino acid enantiomers studied. The amount of D-Asp significantly increased in AtDAAT-deficient A. thaliana, whereas the amounts of the D-Glu, D-Ala, and D-Val decreased significantly, indicating the physiological role of AtDAAT in metabolizing exogenous D-Asp. The amounts of all L-enantiomers were higher in AtDAAT-deficient A. thaliana than in the wild type, indicating the involvement of other amino acid-metabolizing enzymes. Further investigations focusing on the physiological roles of these enzymes in A. thaliana are to be conducted in our laboratory.