X-ray Studies of Sugar-Modifying Enzymes

描述(申请人提供)：碳水化合物约占地球生物量的50%，它们主要在能量储存、信号传递、分子识别和细胞结构中发挥作用。它们也是许多抗生素、抗真菌、驱虫剂和抗肿瘤药物的功效所必需的。事实上，在自然界中观察到的碳水化合物结构的多样性确实令人惊叹。与这项赠款申请特别相关的是在革兰氏阴性细菌的O-抗原中观察到的二脱氧糖和三脱氧糖。研究表明，这些糖具有高度的免疫原性，在细菌的毒力中发挥作用，并帮助有机体逃避治疗剂。特别是，本提案的重点是生产三种糖所需的生物合成酶：(I)甲壳糖、(Ii)2，3-二乙酰胺-2，3-二脱氧-D-甘露糖酸(D-manNAc3NAcA)和(Iii)军氨酸。例如，在霍乱的病原体霍乱弧菌的O-抗原中观察到了Colitose。一种相当罕见的糖，D-ManNAc3NAcA，是从机会细菌铜绿假单胞菌的B带O抗原和百日咳杆菌的A带三糖中分离出来的，铜绿假单胞菌是医院感染的主要来源，百日咳杆菌是引起严重咳嗽的病原体。第三种糖，军团氨基酸，是在嗜肺军团菌中首次发现的，军团菌是导致军团病的细菌。这三种糖是由一系列耐人寻味的酶合成的，在本提案中将通过X射线结晶学、定点突变实验和动力学分析进行研究。这些生物合成途径中的一些蛋白质最终可能成为抗菌药物设计的靶点。此外，拟议的研究将揭示前所未有的化学，并将为机械酶学提供基础贡献。最后，通过了解要研究的酶的结构和功能，将有可能设计出自然界中以前没有遇到过的独特的碳水化合物部分，这些将对新疗法的开发产生重要的影响。 与公众健康相关：这项应用的目标是研究参与二和三脱氧糖生物合成的酶的结构和功能。这些类型的糖在革兰氏阴性细菌的O抗原中观察到，研究表明这些碳水化合物具有高度的免疫原性，在细菌的毒力中发挥作用，并帮助有机体逃避治疗药物。拟议的研究将揭示前所未有的化学变化，并将为机械酶学提供基础贡献。此外，通过了解要研究的酶的结构和功能，将有可能设计出自然界中以前没有遇到过的独特的碳水化合物部分，这些将对新疗法的开发产生重要的影响。

DESCRIPTION (provided by applicant): Carbohydrates constitute ~50% of the Earth's biomass where they major play roles in energy storage, signaling, molecular recognition, and cell structure. They are also required for the efficacies of many antibiotics, antifungals, anthelmintics, and antitumor agents. Indeed, the diversity of carbohydrate structures observed in nature is truly remarkable. Of particular relevance to this grant application are the di- and trideoxysugars observed in the O-antigens of Gram-negative bacteria. Studies have shown that these sugars are highly immunogenic, play roles in the virulence of the bacterium, and help the organism to evade therapeutic agents. In particular, the focus of this proposal is on the biosynthetic enzymes required for the production of three sugars: (i) colitose, (ii) 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid (D- ManNAc3NAcA) and (iii) legionaminic acid. Colitose has been observed, for example, in the O-antigen of Vibrio cholerae, the causative agent of cholera. The quite rare sugar, D-ManNAc3NAcA, has been isolated from the B-band O-antigen of the opportunistic bacterium Pseudomonas aeruginosa, a major source of nosocomial infections and in the A-band trisaccharide of the bacterium Bordetella pertussis, the causative agent of whopping cough. The third sugar, legionaminic acid, was first identified in Legionella pneumophila, the bacterium responsible for Legionnaires' disease. These three sugars are synthesized by an array of intriguing enzymes that will be investigated in this proposal by X-ray crystallography, site-directed mutagenesis experiments, and kinetic analyses. Some of the proteins in these biosynthetic pathways may ultimately serve as targets for antimicrobial drug design. In addition, the proposed investigations will reveal unprecedented chemistries and will provide fundamental contributions to mechanistic enzymology. Finally, by understanding the structures and functions of the enzymes to be investigated, it will be possible to design unique carbohydrate moieties not previously encountered in nature, and these will have important ramifications for the development of new therapeutics. PUBLIC HEALTH RELEVANCE: The goal of this application is to investigate the structures and functions of enzymes involved in the biosynthesis of di- and trideoxysugars. These types of sugars are observed in the O-antigens of Gram-negative bacteria, and studies have shown that these carbohydrates are highly immunogenic, play roles in the virulence of the bacterium, and help the organism to evade therapeutic agents. The proposed investigations will reveal unprecedented chemistries and will provide fundamental contributions to mechanistic enzymology. In addition, by understanding the structures and functions of the enzymes to be investigated, it will be possible to design unique carbohydrate moieties not previously encountered in nature, and these will have important ramifications for the development of new therapeutics.