Mechanistic and structural studies of eukaryotic UDP-galactopyranose mutases

性状（由申请方提供）：呋喃半乳糖（Galf）是致病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缀合结构（糖衣）的主要组分。由于含Galf的分子对于宿主特异性细胞识别、生长和发病机制的重要性，以及在人类中不存在这种不寻常的糖，Galf生物合成酶是开发新的抗微生物剂的有吸引力的靶点。黄素酶UDP-吡喃半乳糖苷酶（UGM）通过催化UDP-吡喃半乳糖转化为UDP-半乳糖醛酸（UDP-Galf）在半乳糖醛酸生物合成中起核心作用。UGM基因的缺失导致真菌病原体烟曲霉和原生动物寄生虫利什曼原虫的毒力严重减弱，这表明UGM是一个有前途的药物设计靶点。此外，UGM从根本上是有趣的，因为该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。在此，我们首次利用A. fumigatus和Trypanosoma cruzi分别作为真菌和原生动物寄生虫的原型。关键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UGM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，表征的氧化还原电位和pH值的档案，测试潜在的替代基板和抑制剂，并确定氧化还原伙伴。2.确定真核UGM的三维结构。将使用X射线晶体学和小角X射线散射来解决氧化、还原和配体结合构象中的UGM结构。这些目标的成功完成将为未来设计基于结构和机制的UGM抑制剂提供平台，其可以作为开发用于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。) 公共卫生相关性：UDP-吡喃半乳糖苷酶（UGM）催化呋喃半乳糖（Galf）生物合成的关键步骤，Galf是病原真菌和致病原生动物寄生虫中发现的必需糖分子。这项研究将为真菌烟曲霉和恰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创造新的知识。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。

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.