Elucidation of D-alanine biosynthetic pathways in the hyperthermophiles: Characterization of alanine racemase from Thermus thermophilus and the structure and dual physiological functions of lysine racemase from Thermotoga maritima.

The hyperthermophilic bacterium Thermotoga maritima contains a peptidoglycan comprising D-alanine, D-glutamate, and D-lysine. We have previously identified the biosynthetic pathways of D-lysine and D-glutamate; however, the biosynthetic pathway of D-alanine is unclear in T. maritima. The present study elucidated the D-alanine biosynthetic pathway in T. maritima, following the characterization of the TTHA0062 activity, constructed a TTHA0062 (encoding alanine racemase)-deficient Thermus thermophilus (ΔTTHA0062) strain. TTHA0062 exhibited the racemase activity toward 10 amino acids including Ala, and the catalytic efficiency of L-alanine was 2-fold higher than that of D-alanine. Regarding the T. maritima genes introduced into the genome of the ΔTTHA0062 strain, TM1597 (lysine racemase) completely restored the growth, TM1270 (multifunctional enzyme) resulted in partial restoration, whereas TM0831 (D-amino acid aminotransferase) had no effect, suggesting that TM1597 and TM1270 are involved in D-alanine biosynthesis in T. maritima. Furthermore, we determined the crystal structure of TM1597 in the internal aldimine form, in which pyridoxal 5'-phosphate (PLP) forms a Schiff base linkage with the active site residue Lys36, and revealed the residues crucial for the catalytic reaction by TM1597 mutational analysis.