D-alanine:D-alanine ligase (EC 18.104.22.168, Ddl) catalyzes the ATP driven ligation of two D-alanine (D-ala) molecules to form the D-alanyl:D-alanine dipeptide. This molecule is a key building block in peptidoglycan biosynthesis making Ddl an attractive target for drug development. D-cycloserine (DCS), an analog of D-ala and a prototype Ddl inhibitor, has shown promise for the treatment of tuberculosis. Here, we report the crystal structure of the Mycobacterium tuberculosis Ddl at a resolution of 2.1 A. This structure indicates that Ddl is a dimer and consists of three discrete domains; the ligand binding cavity is at the intersection of all three domains and conjoined by several loop regions. The M. tuberculosis apo Ddl structure shows a novel conformation that has not yet been observed in Ddl enzymes from other species. The nucleotide and D-alanine binding pockets are flexible, requiring significant structural rearrangement of the bordering regions for entry and binding of both ATP and D-ala molecules. Solution affinity and kinetic studies showed that DCS interacts with Ddl in a manner similar to D-ala. Each ligand binds to two binding sites that have significant differences in affinity with the first binding site exhibiting high affinity. DCS inhibits the enzyme with an IC50 of 0.37 mM under standard assay conditions, implicating a preferential and weak inhibition at the second, lower affinity, binding site. Moreover, DCS binding is tighter at higher ATP concentrations. The crystal structure illustrates potential drugable sites that may result in the development of more effective Ddl inhibitors.
Structure of the Mycobacterium tuberculosis D-alanine:D-alanine Ligase: a target of the anti-tuberculosis drug D-cycloserine.,Bruning JB, Murillo AC, Chacon O, Barletta RG, Sacchettini JC Antimicrob Agents Chemother. 2010 Oct 18. PMID:20956591
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
↑ Bruning JB, Murillo AC, Chacon O, Barletta RG, Sacchettini JC. Structure of the Mycobacterium tuberculosis D-alanine:D-alanine Ligase: a target of the anti-tuberculosis drug D-cycloserine. Antimicrob Agents Chemother. 2010 Oct 18. PMID:20956591 doi:10.1128/AAC.00558-10