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Biochemical Characterization of Specificity for Family 3 Glycoside Hydrolases

家族 3 糖苷水解酶特异性的生化表征

基本信息

批准号：
8387025
负责人：
Dylan Dodd
金额：
$ 2.98万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-16 至 2013-05-04
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8387025
关键词：
Amino Acid Substitution Amino Acids Bacteria Biochemical Carbohydrates Cell Wall Characteristics Colon Crohn&apos s disease DNA Sequence Data Development Diet Dietary Polysaccharide Disease Distal Enzymes Epithelial Evolution Family Fermentation Funding Gastrointestinal Diseases Gene Family Genes Genome Glycoside Hydrolases Goals Health Human Human Microbiome Immune system Libraries Metabolic Metagenomics Microbe Modeling Molecular Nutrient Oligosaccharides Plants Prevotella Property Research Role Rumen Sampling Specificity Substrate Specificity Systems Development Ulcerative Colitis United States National Institutes of Health depolymerization directed evolution effective therapy glucosidase gut microflora human subject insight member microbial microbial community microbial genome microbiome microorganism public health relevance scaffold

项目摘要

DESCRIPTION (provided by applicant): The human distal gut microbiome is an extraordinary example of a mutualistic relationship wherein trillions of microbes ferment dietary and host-derived carbohydrates and the products of fermentation contribute to nutrient acquisition, gut epithelial health, and immune system development. The NIH-funded human microbiome project will generate nearly 1000 reference genomes from cultured and non-cultured microbes, and will supplement this data with DNA sequencing of microbial samples taken from human subjects. Glycoside hydrolases (GHs) comprise a significant proportion of the genes encoded by microbial genomes within the human distal gut microbiome and contribute to the depolymerization of recalcitrant dietary polysaccharides. A recent metagenomic analysis of the human distal gut microbiome revealed that of the 81 GH families present in the distal gut microbiome, GH family 3 was the most highly represented, which indicates that this gene family is important for carbohydrate utilization by the gut microflora. A number of different functional activities have been described for GH family 3 enzymes although the molecular determinants that define substrate specificity for these enzymes have not been elucidated. The long-term goal of the proposed research is to provide insight into the role of the GH family 3 genes in the metabolic repertoire of human gut microorganisms. In aim 1 we will characterize the substrate specificities of four GH family 3 enzymes from the bacterium Prevotella bryantii Bi4 using a library of natural plant cell wall derived oligosaccharides. In aim 2 we will employ a directed evolution approach for identifying amino acid residues that contribute to substrate specificity for one of these GH family 3 enzymes from P. bryantii Bi4. Results from the proposed studies will provide insight into the molecular determinants of substrate specificity for GH family 3 genes and will allow us to place this important gene family in the context of the metabolic repertoire of the gut-associated microflora.

描述（由申请人提供）：人类远端肠道微生物组是互利关系的一个非凡例子，其中数万亿微生物发酵饮食和宿主来源的碳水化合物，发酵产物有助于营养获取、肠道上皮健康和免疫系统发育。美国国立卫生研究院 (NIH) 资助的人类微生物组项目将从培养和非培养的微生物中生成近 1000 个参考基因组，并将通过对取自人类受试者的微生物样本进行 DNA 测序来补充这些数据。糖苷水解酶 (GH) 占人类远端肠道微生物组内微生物基因组编码的基因的很大一部分，有助于顽固膳食多糖的解聚。最近对人类远端肠道微生物组的宏基因组分析显示，在远端肠道微生物组中存在的 81 个 GH 家族中，GH 家族 3 的代表性最高，这表明该基因家族对于肠道微生物群的碳水化合物利用非常重要。尽管尚未阐明定义这些酶的底物特异性的分子决定因素，但已经描述了 GH 家族 3 酶的许多不同功能活性。该研究的长期目标是深入了解 GH 家族 3 基因在人类肠道微生物代谢谱中的作用。在目标 1 中，我们将使用天然植物细胞壁来源的寡糖文库来表征来自布氏普氏菌 Bi4 的四种 GH 家族 3 酶的底物特异性。在目标 2 中，我们将采用定向进化方法来鉴定有助于来自 P. bryantii Bi4 的 GH 家族 3 酶之一的底物特异性的氨基酸残基。拟议研究的结果将提供对 GH 家族 3 基因底物特异性的分子决定因素的深入了解，并使我们能够将这个重要的基因家族置于肠道相关微生物群代谢库的背景下。