title: Structural and functional characterization of the alanine racemase from Streptomyces coelicolor A3(2).
abstract: 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-1for the racemization of L- to D-Ala, and 104 ± 7 U mg-1for 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.
authors.affiliation: Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Tassoni
authors.affiliation: Microbial Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands.
authors.forename: Lizah T
authors.initials: LT
authors.lastname: van der Aart
authors.affiliation: Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden, The Netherlands. Electronic address: m.ubbink@chem.leidenuniv.nl.
authors.forename: Marcellus
authors.initials: M
authors.lastname: Ubbink
authors.affiliation: Microbial Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands. Electronic address: g.wezel@biology.leidenuniv.nl.
authors.forename: Gilles P
authors.initials: GP
authors.lastname: van Wezel
authors.affiliation: Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden, The Netherlands. Electronic address: raj@chem.leidenuniv.nl.
authors.forename: Navraj S
authors.initials: NS
authors.lastname: Pannu
mesh_terms: D000411:Alanine Racemase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003523:Cycloserine / Q000494:pharmacology; D024881:Drug Resistance, Bacterial; D017353:Gene Deletion; D005798:Genes, Bacterial; D007700:Kinetics; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D017386:Sequence Homology, Amino Acid; D048372:Streptomyces coelicolor / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D003523:Cycloserine; D000411:Alanine Racemase
keywords: Actinobacteria; Alanine racemase; Antibiotic resistance; Peptidoglycan; X-ray crystallography