Enzymatic properties and physiological function of glutamate racemase from Thermus thermophilus

Biochim Biophys Acta Proteins Proteom. 2020 Sep;1868(9):140461. doi: 10.1016/j.bbapap.2020.140461. Epub 2020 May 28.


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 (kcat/Km) of TTHA1643 toward d- and l-Glu was comparable; however, the kcat 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.

Keywords: Amino acid racemase; Glutamate racemase; Peptidoglycan; Thermus thermophilus; d-amino acid; d-glutamate.

MeSH terms

  • Amino Acid Isomerases / chemistry*
  • Amino Acid Isomerases / genetics*
  • Amino Acid Isomerases / metabolism*
  • Amino Acid Sequence
  • Amino Acids / metabolism
  • Cell Wall / chemistry
  • Cloning, Molecular
  • Enzyme Stability
  • Escherichia coli / metabolism
  • Gene Deletion
  • Genome, Bacterial
  • Glutamic Acid / metabolism
  • Hydrogen-Ion Concentration
  • Kinetics
  • Recombinant Proteins
  • Substrate Specificity
  • Temperature
  • Thermus thermophilus / enzymology*
  • Thermus thermophilus / genetics
  • Thermus thermophilus / growth & development
  • Thermus thermophilus / physiology
  • Transcriptome


  • Amino Acids
  • Recombinant Proteins
  • Glutamic Acid
  • Amino Acid Isomerases
  • glutamate racemase