X-ray Studies of Sugar-Modifying Enzymes

描述（由申请人提供）：碳水化合物占地球生物量的50%左右，在能量储存、信号传导、分子识别和细胞结构等方面发挥着重要作用。它们也是许多抗生素、抗真菌药、驱虫药和抗肿瘤药的功效所必需的。的确，在自然界中观察到的碳水化合物结构的多样性是非常显著的。与此拨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，并帮助生物体逃避治疗剂。特别是，本提案的重点是生产三种糖所需的生物合成酶：(i)粘胶糖，（ii） 2,3-二乙酰氨基-2,3-二脱氧-D-甘露糖醛酸（D- ManNAc3NAcA）和（iii）军团胺酸。例如，在霍乱病原体霍乱弧菌的o型抗原中已观察到结肠炎。这种非常罕见的糖D-ManNAc3NAcA已经从机会性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，铜绿假单胞菌是医院感染的主要来源，百日咳杆菌是咳嗽的病原体。第三种糖是军团胺酸，最早是在嗜肺军团菌中发现的，这种细菌会导致军团病。这三种糖是由一系列有趣的酶合成的，本文将通过x射线晶体学、定点诱变实验和动力学分析来研究这些酶。这些生物合成途径中的一些蛋白质可能最终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，并将为机制酶学提供基础贡献。最后，通过了解要研究的酶的结构和功能，将有可能设计出以前在自然界中没有遇到过的独特的碳水化合物部分，这些将对新疗法的发展产生重要影响。

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.