TY - JOUR T1 - Structural and Functional Characterization of the Alanine Racemase from <em>Streptomyces coelicolor</em> A3(2) JF - bioRxiv DO - 10.1101/089938 SP - 089938 AU - Raffaella Tassoni AU - L.T. van der Aart AU - M. Ubbink AU - G. P. Van Wezel AU - N. S. Pannu Y1 - 2016/01/01 UR - http://biorxiv.org/content/early/2016/12/20/089938.abstract N2 - 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. Specificity 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. ER -