D-alanine:D-alanine ligase (EC 6.3.2.4; 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 Mycobacterium tuberculosis Ddl at a resolution of 2.1 Å. 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 that observed for 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 a 50% inhibitory concentration (IC(50)) 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.