PT  - JOURNAL ARTICLE
AU  - Raffaella Tassoni
AU  - L.T. van der Aart
AU  - M. Ubbink
AU  - G.P. Van Wezel
AU  - N.S. Pannu
TI  - Structural and Functional Characterization of the Alanine Racemase from <em>Streptomyces coelicolor</em> A3(2)
AID  - 10.1101/089938
DP  - 2016 Jan 01
TA  - bioRxiv
PG  - 089938
4099  - http://biorxiv.org/content/early/2016/11/26/089938.short
4100  - http://biorxiv.org/content/early/2016/11/26/089938.full
AB  - 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. 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.