Enzymatic properties and physiological function of glutamate racemase from Thermus thermophilus

D-Amino acids are physiologically important components of peptidoglycan in the bacterial cell wall, maintaining cell structure and aiding adaptation to environmental changes through peptidoglycan remodelling. Therefore, the biosynthesis of D-amino acids is essential for bacteria to adapt to different environmental conditions. The peptidoglycan of the extremely thermophilic bacterium Thermus thermophilus contains D-alanine (D-Ala) and D-glutamate (D-Glu), but its D-amino acid metabolism remains poorly understood. Here, we investigated the enzyme activity and function of the product of the TTHA1643 gene, which is annotated to be a Glu racemase in the T. thermophilus HB8 genome. Among 21 amino acids tested, TTHA1643 showed highly specific activity toward Glu as the substrate. The catalytic efficiency (k(cat)/K-m) of TTHA1643 toward D- and L-Glu was comparable; however, the k(cat) value was 18-fold higher for L-Glu than for D-Glu. Temperature and pH profiles showed that the racemase activity of TTHA1643 is high under physiological conditions for T. thermophilus growth. To assess physiological relevance, we constructed a TTHA1643-deficient strain (Delta TTHA1643) by replacing the TTHA1643 gene with the thermostable hygromycin resistance gene. Growth of the Delta TTHA1643 strain in synthetic medium without D-Glu was clearly diminished relative to wild type, although the TTHA1643 deletion was not lethal, suggesting that alternative D-Glu biosynthetic pathways may exist. The deterioration in growth was restored by adding D-Glu to the culture medium, showing that D-Glu is required for normal growth of T. thermophilus. Collectively, our findings show that TTHA1643 is a Glu racemase and has the physiological function of D-Glu production in T. thermophilus.