Structure of the Mycobacterium tuberculosis D-Alanine:D-Alanine Ligase, a Target of the Antituberculosis Drug D-Cycloserine
Structure of the Mycobacterium tuberculosis D-Alanine:D-Alanine Ligase, a Target of the Antituberculosis Drug D-Cycloserine
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DOI：
10.1128/aac.00558-10
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发表时间：
2011-01-01
影响因子：
4.9
通讯作者：
Sacchettini, James C.
中科院分区：
文献类型：
--
作者：
Bruning, John B.;Murillo, Ana C.;Sacchettini, James C.
D-丙氨酸：D-丙氨酸连接酶（EC 6.3.2.4; Ddl）催化两个D-丙氨酸（D-Ala）分子的ATP驱动的连接以形成D-丙氨酰：D-丙氨酸二肽。该分子是肽聚糖生物合成中的关键构件，使得Ddl成为药物开发的有吸引力的靶标。D-环丝氨酸（DCS），D-丙氨酸的类似物和原型Ddl抑制剂，已显示出用于治疗结核病的前景。在这里，我们报告结核分枝杆菌Ddl的晶体结构，分辨率为2.1埃。该结构表明Ddl是二聚体并且由三个离散结构域组成;配体结合腔位于所有三个结构域的交叉点处并且由几个环区域连接。分枝结核病载脂蛋白Ddl结构显示了在来自其他物种的Ddl酶中尚未观察到的新构象。核苷酸和D-丙氨酸结合口袋是灵活的，需要边界区域的显著结构重排以进入和结合ATP和D-Ala分子。溶液亲和性和动力学研究表明，DCS与Ddl的相互作用的方式类似的D-Ala所观察到的。每个配体结合两个亲和力显着差异的结合位点，其中第一个结合位点表现出高亲和力。DCS抑制酶，在标准测定条件下具有0.37 mM的50%抑制浓度（IC 50），这意味着在第二个较低亲和力结合位点处具有优先和弱抑制作用。此外，DCS结合在较高的ATP浓度下更紧密。晶体结构说明了可能导致开发更有效的Ddl抑制剂的潜在药物位点。
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 angstrom. 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.