Mechanistic and structural studies of eukaryotic UDP-galactopyranose mutases

描述（由申请人提供）：半乳糖呋喃糖（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结合结构（糖衣）的主要成分。由于含半胱氨酸分子对宿主特异性细胞识别、生长和发病机制的重要性，以及人类中不存在这种不寻常的糖，半胱氨酸生物合成酶是开发新的抗菌药物的有吸引力的靶标。黄酶udp -半乳糖酰脲酶（UGM）通过催化udp -半乳糖酰脲向udp -半乳糖酰脲的转化，在半乳糖酰脲的生物合成中起着核心作用。UGM基因的缺失导致真菌病原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是一个有前景的药物设计靶点。此外，UGM从根本上是有趣的，因为这种酶既不氧化也不还原底物，这在黄酶中是不寻常的。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，我们首次提出了真核UGMs的催化机制和三维结构的研究，分别使用烟曲霉和克氏锥虫的酶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产生和初步晶体的生长。提出了两个目标：1。确定黄素辅助因子在真核UGMs化学机制中的作用。提出的实验包括快速反应动力学光谱分析，氧化还原电位和pH谱的表征，测试潜在的替代底物和抑制剂，以及识别氧化还原伙伴。2. 确定真核生物UGMs的三维结构。利用x射线晶体学和小角x射线散射，可以解析氧化、还原和配体结合构象的UGMs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UGMs抑制剂提供一个平台，这些抑制剂可以作为开发用于治疗真菌感染和被忽视疾病（如恰加斯病）的化疗药物的先导化合物。

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.