Biochemical Characterization of Specificity for Family 3 Glycoside Hydrolases

描述(由申请人提供)： 人类远端肠道微生物群是一种互惠互利关系的非同寻常的例子，在这种关系中，数万亿微生物发酵膳食和宿主衍生的碳水化合物以及发酵产物，有助于营养获取、肠道上皮健康和免疫系统发育。由美国国立卫生研究院资助的人类微生物组项目将从培养的和非培养的微生物中产生近1000个参考基因组，并将用从人类受试者身上采集的微生物样本的DNA测序来补充这些数据。糖苷水解酶(GHS)是人体远端肠道微生物组中微生物基因组编码的基因的重要组成部分，对顽固性膳食多糖的解聚起重要作用。最近对人类远端肠道微生物组的元基因组分析表明，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肠道微生物区系对碳水化合物的利用具有重要作用。生长激素家族3的许多不同的功能活性已经被描述，尽管定义这些酶底物特异性的分子决定因素还没有被阐明。这项拟议研究的长期目标是深入了解生长激素家族3基因在人类肠道微生物代谢过程中的作用。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细菌Prevotea bryantii Bi4的四种生长激素3酶的底物特异性。在目标2中，我们将使用定向进化的方法来鉴定与来自P.bryantii Bi4的GH家族3酶之一底物专一性有关的氨基酸残基。拟议的研究结果将为我们深入了解GH家族3基因底物特异性的分子决定因素，并将使我们能够将这个重要的基因家族置于肠道相关微生物区系的代谢谱系的背景下。 公共卫生相关性：人体结肠内微生物群落的组成是一个关键 影响多种疾病状态发展的因素，包括克罗恩病和 溃疡性结肠炎。这项研究将对人类结肠的代谢特性提供直接的见解 微生物区系。了解肠道微生物区系的代谢特征是有效的 治疗各种胃肠道疾病。

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 neariy 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 farnily 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. Public Health Relevance: The composition of microbial communities within the human colon is a critical factor that influences the development of numerous disease states including Crohn's disease and ulcerative colitis. This study will provide direct insight into the metabolic properties of the human colonic microflora. Understanding the metabolic characteristics of the gut microflora is essential for effective treatment of a wide variety of gastrointestinal disorders.