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 (∆TTHA1643) by replacing the TTHA1643 gene with the thermostable hygromycin resistance gene. Growth of the ∆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.

d-氨基酸是肽聚糖在细菌细胞壁中的重要生理成分，可维持细胞结构并通过肽聚糖重塑帮助适应环境变化。因此，d-氨基酸的生物合成对于细菌适应不同的环境条件是必不可少的。肽聚糖的极端嗜热菌嗜热栖热包含d丙氨酸（d -Ala）和d -谷氨酸（d -Glu），但它的d -氨基酸代谢仍然知之甚少。在这里，我们研究了TTHA1643产品的酶活性和功能该基因被注释为嗜热嗜热菌HB8基因组中的Glu消旋酶。在测试的21个氨基酸中，TTHA1643对Glu作为底物表现出高度的比活性。TTHA1643对d-和l -Glu的催化效率（k _cat / K _m）相当; 然而，l -Glu的k _cat值比d -Glu高18倍。温度和pH曲线表明，在生理条件下，TTHA1643的消旋酶活性较高，可促进嗜热链球菌的生长。为了评估生理相关性，我们构建了TTHA1643通过用热稳定的潮霉素抗性基因替代TTHA1643基因，从而获得低毒菌株（∆TTHA1643）。相对于野生型，ΔTTHA1643菌株在无d -Glu的合成培养基中的生长明显减少，尽管TTHA1643缺失并不致命，这表明可能存在替代的d -Glu生物合成途径。通过向培养基中添加d -Glu来恢复生长的恶化，这表明d -Glu是嗜热链球菌正常生长所必需的。总的来说，我们的发现表明TTHA1643是一种Glu消旋酶，具有d的生理功能。-嗜热链球菌中的-Glu生产。