Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri 65211, USA.
Biochemistry. 2012 Jun 19;51(24):4968-79. doi: 10.1021/bi300498c. Epub 2012 Jun 5.
Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitor design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3 Å movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45% identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10-50, primarily by decreasing k(cat). Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens.
恰加斯病是一种被忽视的热带病，由原生动物寄生虫克氏锥虫引起。在这里，我们报告了克氏锥虫半乳糖呋喃糖生物合成酶 UDP-半乳糖吡喃糖变位酶（UGM）的晶体结构，这是该酶来自原生动物寄生虫的第一个结构。UGM 是药物设计的一个有吸引力的靶点，因为半乳糖呋喃糖在人类中不存在，但它是锥虫生物中关键糖蛋白和糖脂的重要组成部分。对酶-UDP 非共价相互作用和序列比对的分析表明，底物识别在真核 UGM 中高度保守，与细菌 UGM 不同。这一观察结果对抑制剂设计具有重要意义。通过还原 FAD 激活酶会引起深刻的构象变化，包括组氨酸环（Gly60-Gly61-His62）的 2.3 Å 移动、His62 的咪唑的旋转和质子化以及位于 FAD si 面的残基的协同运动。有趣的是，这些变化与曲霉 UGM 描述的变化有很大不同，曲霉 UGM 与 T. cruzi UGM 的同一性为 45%。用定点突变酶 G61A、G61P 和 H62A 研究了 Gly61 和 His62 对酶活性的重要性。这些突变使催化效率降低了 10-50 倍，主要是通过降低 k(cat)。综合来看，结构、动力学和序列数据表明，组氨酸环的中间 Gly 赋予了真核 UGM 激活所必需的灵活性。我们的结果提供了关于 UGM 生物化学的新信息，并提出了一种从真核病原体设计 UGM 抑制剂的统一策略。
