Pegan Scott D, Rukseree Kamolchanok, Franzblau Scott G, Mesecar Andrew D
Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, IL 60607, USA.
J Mol Biol. 2009 Mar 6;386(4):1038-53. doi: 10.1016/j.jmb.2009.01.003. Epub 2009 Jan 10.
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), currently infects one-third of the world's population in its latent form. The emergence of multidrug-resistant and extensive drug-resistant strains has highlighted the need for new pharmacological targets within M. tuberculosis. The class IIa fructose 1,6-bisphosphate aldolase (FBA) enzyme from M. tuberculosis (MtFBA) has been proposed as one such target since it is upregulated in latent TB. Since the structure of MtFBA has not been determined and there is little information available on its reaction mechanism, we sought to determine the X-ray structure of MtFBA in complex with its substrates. By lowering the pH of the enzyme in the crystalline state, we were able to determine a series of high-resolution X-ray structures of MtFBA bound to dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and fructose 1,6-bisphosphate at 1.5, 2.1, and 1.3 A, respectively. Through these structures, it was discovered that MtFBA belongs to a novel tetrameric class of type IIa FBAs. The molecular details at the interface of the tetramer revealed important information for better predictability of the quaternary structures among the FBAs based on their primary sequences. These X-ray structures also provide interesting and new details on the reaction mechanism of class II FBAs. Substrates and products were observed in geometries poised for catalysis; in addition, unexpectedly, the hydroxyl-enolate intermediate of dihydroxyacetone phosphate was also captured and resolved structurally. These concise new details offer a better understanding of the reaction mechanisms for FBAs in general and provide a structural basis for inhibitor design efforts aimed at this class of enzymes.
结核分枝杆菌是结核病（TB）的病原体，目前以潜伏形式感染着全球三分之一的人口。多重耐药和广泛耐药菌株的出现凸显了在结核分枝杆菌中寻找新的药理学靶点的必要性。结核分枝杆菌的IIa类果糖1,6-二磷酸醛缩酶（FBA）（MtFBA）被认为是这样一个靶点，因为它在潜伏性结核病中表达上调。由于MtFBA的结构尚未确定，且关于其反应机制的信息很少，我们试图确定与底物结合的MtFBA的X射线结构。通过降低晶体状态下酶的pH值，我们分别以1.5 Å、2.1 Å和1.3 Å的分辨率确定了一系列与磷酸二羟丙酮、3-磷酸甘油醛和1,6-二磷酸果糖结合的MtFBA的高分辨率X射线结构。通过这些结构发现，MtFBA属于IIa类FBA的一种新型四聚体类型。四聚体界面处的分子细节揭示了基于其一级序列更好地预测FBA中四级结构的重要信息。这些X射线结构还为II类FBA的反应机制提供了有趣的新细节。观察到底物和产物处于有利于催化的几何构型；此外，出乎意料的是，磷酸二羟丙酮的羟基烯醇中间体也被捕获并解析了其结构。这些简洁的新细节有助于更好地理解一般FBA的反应机制，并为针对这类酶的抑制剂设计努力提供了结构基础。
