Structure of the Mycobacterium tuberculosis D-alanine:D-alanine ligase, a target of the antituberculosis drug D-cycloserine

John B. Bruning, Ana C. Murillo, Ofelia Chacon, Raul G Barletta, James C. Sacchettini

Research output: Contribution to journalArticle

61 Citations (Scopus)

Abstract

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 (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.

Original languageEnglish (US)
Pages (from-to)291-301
Number of pages11
JournalAntimicrobial Agents and Chemotherapy
Volume55
Issue number1
DOIs
StatePublished - Jan 1 2011

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Cycloserine
Mycobacterium tuberculosis
Alanine
Adenosine Triphosphate
Binding Sites
Pharmaceutical Preparations
Inhibitory Concentration 50
Ligands
Peptidoglycan
Dipeptides
Enzymes
Ligation
Tuberculosis
Nucleotides
D-alanylalanine synthetase

ASJC Scopus subject areas

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases

Cite this

Structure of the Mycobacterium tuberculosis D-alanine:D-alanine ligase, a target of the antituberculosis drug D-cycloserine. / Bruning, John B.; Murillo, Ana C.; Chacon, Ofelia; Barletta, Raul G; Sacchettini, James C.

In: Antimicrobial Agents and Chemotherapy, Vol. 55, No. 1, 01.01.2011, p. 291-301.

Research output: Contribution to journalArticle

Bruning, John B. ; Murillo, Ana C. ; Chacon, Ofelia ; Barletta, Raul G ; Sacchettini, James C. / Structure of the Mycobacterium tuberculosis D-alanine:D-alanine ligase, a target of the antituberculosis drug D-cycloserine. In: Antimicrobial Agents and Chemotherapy. 2011 ; Vol. 55, No. 1. pp. 291-301.
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abstract = "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 {\AA}. 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 (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.",
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