Mechanistic and structural studies of eukaryotic UDP-galactopyranose mutases

真核UDP-吡喃半乳糖变位酶的机制和结构研究

• 批准号: 8532685
• 负责人: Pablo Sobrado
• 金额: $ 27.7万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-15 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8532685
• 关键词: Acute; Anabolism; Aspergillus; Aspergillus fumigatus; Attenuated; Bacteria; Binding; Catalysis; Cell Wall; Cell surface; Cells; Chagas Disease; Chemicals; Complement; Complex; Development; Disease; Drug Design; Electron Transport; Enzyme Inhibition; Enzymes; Flavins; Future; Galactose; Genes; Genus Mycobacterium; Goals; Growth; Human; Kinetics; Knowledge; Lead; Leishmania; Leishmania major; Ligand Binding; Measures; Molecular Conformation; Mycoses; Nature; Outcome; Oxidation-Reduction; Oxidoreductase; Parasites; Pathogenesis; Play; Production; Reaction; Recombinants; Research; Roentgen Rays; Role; Solutions; Structure; Testing; Trypanosoma cruzi; UDP-galactopyranose mutase; Virulence; Virulence Factors; X-Ray Crystallography; analog; antimicrobial drug; base; cofactor; design; enzyme structure; fungus; inhibitor/antagonist; interest; neglect; novel; pathogen; prevent; prototype; public health relevance; research study; small molecule; sugar; three dimensional structure; uridine diphosphate galactofuranose

DESCRIPTION (provided by applicant): Galactofuranose (Galf) is an important building block of the cell wall of pathogenic fungi and a major component of the cell surface glycoconjugated structures (sugar coat) of protozoan parasites. Because of the importance of Galf-containing molecules for host-specific cell recognition, growth, and pathogenesis, and the absence of this unusual sugar in humans, Galf biosynthetic enzymes are attractive targets for the development of new antimicrobial agents. The flavoenzyme UDP-galactopyranose mutase (UGM) plays a central role in Galf biosynthesis by catalyzing the conversion of UDP-galactopyranose to UDP-Galf. Deletion of the UGM gene results in severely attenuated virulence of the fungal pathogen Aspergillus fumigatus and the protozoan parasite Leishmania major, suggesting UGM as a promising drug design target. In addition, UGM is fundamentally interesting because the enzyme neither oxidizes nor reduces the substrate, which is unusual among flavoenzymes. Studies of bacterial UGMs have shown that the reduced flavin is necessary for catalysis, but the role that the flavin plays during the catalytic cycle remains controversial. Here, we propose the first studies of the catalytic mechanism and three-dimensional structure of eukaryotic UGMs, using the enzymes from A. fumigatus and Trypanosoma cruzi as prototypes from fungal and protozoan parasites, respectively. Key preliminary results include the production of active recombinant enzyme and the growth of preliminary crystals. Two aims are proposed: 1. Determine the role of the flavin cofactor in the chemical mechanism of eukaryotic UGMs. Experiments proposed include rapid reaction kinetic spectroscopic analyses, characterization of the redox potential and pH profiles, testing potential alternative substrates and inhibitors, and identifying redox partners. 2. Determine the three-dimensional structures of eukaryotic UGMs. Structures of UGMs in the oxidized, reduced, and ligand-bound conformations will be solved using X-ray crystallography and small-angle X-ray scattering. Successful completion of these aims will provide a platform for the future design of structure- and mechanism- based inhibitors of UGMs, which could serve as lead compounds for the development of chemotherapeutics for the treatment of fungal infections and neglected diseases such as Chagas disease. ) PUBLIC HEALTH RELEVANCE: UDP-galactopyranose mutase (UGM) catalyzes a key step in the biosynthesis of galactofuranose (Galf), an essential sugar molecule found in pathogenic fungi and disease-causing protozoan parasites. The proposed research will create new knowledge about the catalytic mechanism and three-dimensional structure of UGM from the fungus Aspergillus fumigatus and the causative agent of Chagas disease, the parasite Trypanosoma cruzi. This information will aid the development of antimicrobial agents targeting these important human pathogens.

描述(申请人提供)：半乳呋喃糖(Galf)是病原真菌细胞壁的重要组成部分，也是原生动物寄生虫细胞表面糖共轭结构(糖衣)的主要成分。由于含有GALF的分子对宿主特异性细胞识别、生长和致病的重要性，以及人类体内不存在这种不寻常的糖，GALF生物合成酶是开发新的抗菌剂的有吸引力的靶标。黄素酶UDP-半乳糖变位酶(UGM)通过催化UDP-半乳糖转化为UDP-GALF，在GALF的生物合成中起着核心作用。UGM基因的缺失导致真菌病原菌烟曲霉和原虫寄生虫利什曼原虫的毒力严重减弱，这表明UGM是一种有前途的药物设计靶点。此外，UGM从根本上来说很有趣，因为这种酶既不氧化也不还原底物，这在黄素酶中是不寻常的。对细菌UGM的研究表明，还原的黄素是催化所必需的，但黄素在催化循环中所起的作用仍然存在争议。在这里，我们首次以烟曲霉和克氏锥虫的酶分别作为真菌和原生动物寄生虫的原型，对真核生物UGM的催化机制和三维结构进行了研究。主要的初步结果包括活性重组酶的生产和初步晶体的生长。提出了两个目标：1.确定黄素辅因子在真核生物超微结构化学机制中的作用。建议的实验包括快速反应动力学光谱分析、氧化还原电位和pH曲线的表征、测试潜在的替代底物和抑制剂，以及确定氧化还原伙伴。2.确定真核生物超微结构。氧化、还原和配体结合构象的UGM的结构将用X射线结晶学和小角X射线散射来解决。这些目标的成功完成将为未来基于结构和机制的UGMS抑制剂的设计提供平台，这些抑制剂可能成为开发治疗真菌感染和被忽视的疾病(如恰加斯病)的化疗药物的先导化合物。) 与公共健康相关：UDP-半乳糖变位酶(UGM)催化半乳呋喃糖(GALF)生物合成的关键步骤，GALF是病原真菌和致病原虫寄生虫中发现的一种基本糖分子。这项拟议的研究将创造新的知识，从真菌烟曲霉和查加斯病的病原体，寄生虫克氏锥虫的UGM的催化机制和三维结构。这些信息将有助于开发针对这些重要人类病原体的抗菌剂。