Structural and functional characterization of the alanine racemase from Streptomyces coelicolor A3(2)

Biochem Biophys Res Commun. 2017 Jan 29;483(1):122-128. doi: 10.1016/j.bbrc.2016.12.183. Epub 2016 Dec 29.


The conversion of l-alanine (L-Ala) into d-alanine (D-Ala) in bacteria is performed by pyridoxal phosphate-dependent enzymes called alanine racemases. D-Ala is an essential component of the bacterial peptidoglycan and hence required for survival. The Gram-positive bacterium Streptomyces coelicolor has at least one alanine racemase encoded by alr. Here, we describe an alr deletion mutant of S. coelicolor which depends on D-Ala for growth and shows increased sensitivity to the antibiotic d-cycloserine (DCS). The crystal structure of the alanine racemase (Alr) was solved with and without the inhibitors DCS or propionate, at 1.64 Å and 1.51 Å resolution, respectively. The crystal structures revealed that Alr is a homodimer with residues from both monomers contributing to the active site. The dimeric state of the enzyme in solution was confirmed by gel filtration chromatography, with and without L-Ala or d-cycloserine. The activity of the enzyme was 66 ± 3 U mg-1 for the racemization of L- to D-Ala, and 104 ± 7 U mg-1 for the opposite direction. Comparison of Alr from S. coelicolor with orthologous enzymes from other bacteria, including the closely related d-cycloserine-resistant Alr from S. lavendulae, strongly suggests that structural features such as the hinge angle or the surface area between the monomers do not contribute to d-cycloserine resistance, and the molecular basis for resistance therefore remains elusive.

Keywords: Actinobacteria; Alanine racemase; Antibiotic resistance; Peptidoglycan; X-ray crystallography.

MeSH terms

  • Alanine Racemase / chemistry*
  • Alanine Racemase / genetics
  • Alanine Racemase / metabolism*
  • Amino Acid Sequence
  • Anti-Bacterial Agents / pharmacology
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Catalytic Domain
  • Crystallography, X-Ray
  • Cycloserine / pharmacology
  • Drug Resistance, Bacterial
  • Gene Deletion
  • Genes, Bacterial
  • Kinetics
  • Models, Molecular
  • Protein Structure, Quaternary
  • Sequence Homology, Amino Acid
  • Streptomyces coelicolor / drug effects
  • Streptomyces coelicolor / enzymology*
  • Streptomyces coelicolor / genetics


  • Anti-Bacterial Agents
  • Bacterial Proteins
  • Cycloserine
  • Alanine Racemase