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组是互惠关系的一个非凡例子，其中数万亿微生物发酵膳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。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产生近1000个参考基因组，并将通过对从人类受试者中采集的微生物样本进行DNA测序来补充这些数据。糖苷水解酶（GH）包含由人类远端肠道微生物组内的微生物基因组编码的显著比例的基因，并且有助于柠檬酸膳食多糖的解聚。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肠道微生物菌群的碳水化合物利用很重要。已经描述了GH家族3酶的许多不同的功能活性，尽管定义这些酶的底物特异性的分子决定簇尚未阐明。拟议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衍生的寡糖库来表征来自细菌普氏普雷沃氏菌Bi 4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对来自布氏毕赤酵母Bi 4的这些GH家族3酶之一的底物特异性有贡献的氨基酸残基。从拟议的研究结果将提供深入了解GH家族3基因的底物特异性的分子决定因素，并将使我们能够把这个重要的基因家族的背景下，肠道相关的微生物菌群的代谢库。

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.