Wang Shuishu, Eisenberg David
Public Health Research Institute, 225 Warren Street, Newark, New Jersey 07103, USA.
Biochemistry. 2006 Feb 14;45(6):1554-61. doi: 10.1021/bi051873e.
Pantothenate synthetase (PS) from Mycobacterium tuberculosis represents a potential target for antituberculosis drugs. PS catalyzes the ATP-dependent condensation of pantoate and beta-alanine to form pantothenate. Previously, we determined the crystal structure of PS from M. tuberculosis and its complexes with AMPCPP, pantoate, and pantoyl adenylate. Here, we describe the crystal structure of this enzyme complexed with AMP and its last substrate, beta-alanine, and show that the phosphate group of AMP serves as an anchor for the binding of beta-alanine. This structure confirms that binding of beta-alanine in the active site cavity can occur only after formation of the pantoyl adenylate intermediate. A new crystal form was also obtained; it displays the flexible wall of the active site cavity in a conformation incapable of binding pantoate. Soaking of this crystal form with ATP and pantoate gives a fully occupied complex of PS with ATP. Crystal structures of these complexes with substrates, the reaction intermediate, and the reaction product AMP provide a step-by-step view of the PS-catalyzed reaction. A detailed reaction mechanism and its implications for inhibitor design are discussed.
结核分枝杆菌的泛酸合成酶（PS）是抗结核药物的一个潜在靶点。PS催化泛解酸和β-丙氨酸在ATP依赖下缩合形成泛酸。此前，我们测定了结核分枝杆菌PS的晶体结构及其与AMPCPP、泛解酸和泛酰腺苷酸的复合物结构。在此，我们描述了该酶与AMP及其最后一种底物β-丙氨酸形成的复合物的晶体结构，并表明AMP的磷酸基团作为β-丙氨酸结合的锚定基团。该结构证实，只有在形成泛酰腺苷酸中间体后，β-丙氨酸才能在活性位点腔中结合。还获得了一种新的晶体形式；它展示了活性位点腔的柔性壁处于无法结合泛解酸的构象。用ATP和泛解酸浸泡这种晶体形式可得到PS与ATP完全占据的复合物。这些与底物、反应中间体和反应产物AMP形成的复合物的晶体结构提供了PS催化反应的逐步视图。讨论了详细的反应机制及其对抑制剂设计的意义。
