Structure of the Mycobacterium tuberculosis D-alanine:D-alanine Ligase, a target of the antituberculosis drug D-Cycloserine

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dc.contributor.authorBruning, J.
dc.contributor.authorMurillo, A.
dc.contributor.authorChacon, O.
dc.contributor.authorBarletta, R.
dc.contributor.authorSacchettini, J.
dc.date.issued2011
dc.description.abstractD-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.
dc.description.statementofresponsibilityJohn B. Bruning, Ana C. Murillo, Ofelia Chacon, Raúl G. Barletta and James C. Sacchettini
dc.identifier.citationAntimicrobial Agents and Chemotherapy, 2011; 55(1):291-301
dc.identifier.doi10.1128/AAC.00558-10
dc.identifier.issn0066-4804
dc.identifier.issn1098-6596
dc.identifier.orcidBruning, J. [0000-0002-6919-1824]
dc.identifier.urihttp://hdl.handle.net/2440/74932
dc.language.isoen
dc.publisherAmer Soc Microbiology
dc.rightsCopyright © 2011, American Society for Microbiology. All Rights Reserved.
dc.source.urihttps://doi.org/10.1128/aac.00558-10
dc.subjectMycobacterium tuberculosis
dc.subjectCycloserine
dc.subjectPeptide Synthases
dc.subjectAntitubercular Agents
dc.subjectCalorimetry
dc.subjectMolecular Sequence Data
dc.titleStructure of the Mycobacterium tuberculosis D-alanine:D-alanine Ligase, a target of the antituberculosis drug D-Cycloserine
dc.typeJournal article
pubs.publication-statusPublished

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