X-ray Studies of Sugar-Modifying Enzymes

糖修饰酶的 X 射线研究

• 批准号: 8000156
• 负责人: Hazel M. Holden
• 金额: $ 8.28万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-15 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8000156
• 关键词: Affect; Anabolism; Antibiotics; Antigens; Biochemical; Biological; Biological Process; Carbohydrates; Carbon; Cell Adhesion; Cell Communication; Cells; Chimeric Proteins; Clinical; Defect; Deoxy Sugars; Disease; Enzyme Gene; Enzymes; Erythromycin; Fertilization; Foundations; Funding; Galactose; Galactosemias; Genes; Genetic; Genetic Transcription; Goals; Gram-Negative Bacteria; Grant; Immune response; Impairment; Indium; Investigation; Laboratories; Lead; Leloir Pathway of Galactose Metabolism; Macrolide Antibiotics; Metabolic; Metabolism; Molecular; Nature; O Antigens; Organism; Pathogenicity; Pathway interactions; Physiological Processes; Play; Proteins; Research; Roentgen Rays; Role; Saccharomyces cerevisiae; Severities; Site-Directed Mutagenesis; Source; Structure; System; Techniques; Transcription Coactivator; Transcription Repressor/Corepressor; Transcriptional Regulation; Yeasts; antimicrobial drug; base; colitose; design; desosamine; interest; sugar; tyvelose

DESCRIPTION (provided by applicant): The biological importance of carbohydrates cannot be overstated for they are essential elements in nearly every physiological process and represent the most abundant bio-molecules in living systems. Apart from their role in providing metabolic energy, carbohydrates are involved in a wide range of biological processes including the immune response, cell-cell interactions, fertilization, and cell adhesion, among others. This grant has supported over the last 11 years both x-ray structural and biochemical analyses of the four enzymes that constitute the Leloir pathway for galactose metabolism. Defects in the genes encoding these enzymes can lead to diseased states referred to collectively as galactosemia. Clinical severity of these disorders varies widely, depending upon the gene affected, and the resulting degree of enzyme impairment. Through the efforts of this laboratory, the structures of all of these enzymes are now known, two of which were solved during the current funding period. Additionally, in recent years, this grant has supported the structural analyses of enzymes involved in the synthesis of the unusual deoxysugars: tyvelose, colitose, and desosamine. Tyvelose and colitose are 3,6-dideoxyhexoses that occur in the O-antigens of some Gram- negative bacteria and have been speculated to play a role in the pathogenicity of these organisms. Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found in certain macrolide antibiotics such as the commonly prescribed erythromycin. The addition of unusual deoxysugars such as D-desosamine to such polyketide antibiotics provides or enhances their biological activity. The goals of this competitive renewal are twofold in nature: (i) to continue our research on the structure and function of enzymes involved in deoxysugar biosynthesis, and (ii) to initiate structural studies on the proteins in Saccharomyces cerevisiae that regulate transcription of the genes encoded by the GAL genetic switch. This new study is a natural extension to our past research on the Leloir pathway. Specifically, the GAL genetic switch encodes the genes for the enzymes of the Leloir pathway. Thus far three key components have been identified in controlling transcription of these genes: GalSp, GalSOp, and Gal4p and these will be targeted for structural/functional studies. For all of the planned investigations, a combination of x-ray crystallographic, site-directed mutagenesis, and biochemical techniques will be employed. Taken together, these studies will provide new information with respect to both sugar metabolism and eukaryotic transcriptional regulation.

描述（由申请人提供）：碳水化合物的生物学重要性怎么强调都不为过，因为它们是几乎每个生理过程中的必需元素，并且代表了生命系统中最丰富的生物分子。除了提供代谢能量之外，碳水化合物还参与广泛的生物过程，包括免疫反应、细胞间相互作用、受精和细胞粘附等。这笔资助在过去 11 年中支持了对构成半乳糖代谢 Leloir 途径的四种酶的 X 射线结构和生化分析。编码这些酶的基因缺陷可能导致统称为半乳糖血症的疾病状态。这些疾病的临床严重程度差异很大，具体取决于受影响的基因以及由此产生的酶损伤程度。通过该实验室的努力，所有这些酶的结构现已已知，其中两种在当前资助期间已得到解决。此外，近年来，这笔资助还支持了参与合成不寻常脱氧糖的酶的结构分析：tyvelose、colitose 和 desosamine。 Tyvelose 和 colitose 是 3,6-二脱氧己糖，存在于一些革兰氏阴性细菌的 O 抗原中，推测在这些生物体的致病性中发挥作用。 Desosamine 是一种 3-(二甲氨基)-3,4,6-三脱氧己糖，存在于某些大环内酯类抗生素中，例如常用的红霉素。将不寻常的脱氧糖例如D-去糖胺添加到此类聚酮化合物抗生素中可提供或增强其生物活性。这种竞争性更新的目标本质上是双重的：（i）继续我们对参与脱氧糖生物合成的酶的结构和功能的研究，以及（ii）启动对酿酒酵母中调节基因转录的蛋白质的结构研究由 GAL 基因开关编码。这项新研究是我们过去对勒卢瓦途径研究的自然延伸。具体来说，GAL 基因开关编码 Leloir 途径酶的基因。到目前为止，已经确定了控制这些基因转录的三个关键组件：GalSp、GalSOp 和 Gal4p，这些将成为结构/功能研究的目标。对于所有计划的研究，将结合使用 X 射线晶体学、定点诱变和生化技术。总而言之，这些研究将提供有关糖代谢和真核转录调控的新信息。