Arabidopsis 2010: Collaborative Project on the Functional Genomics of Arabidopsis beta-Glucosidase and beta-Galactosidase Gene Families

O-糖苷水解酶(EC 3.2.1-)广泛存在于动物、植物和微生物中，是催化两种或两种以上碳水化合物之间或碳水化合物与非碳水化合物之间化学键断裂的酶。为了应对2010项目的挑战，即在未来十年内确定所有拟南芥基因的功能，这项合作研究将集中在糖苷水解酶(http://www.biol.vt.edu/faculty/esen/glycosidase.lab.html和http://afmb.cnrs-mrs.fr/~pedro/CAZY/).两个相关家族的大约75个成员家族1包括β-葡萄糖苷酶(EC 3.2.1.21)和芥子酶(EC 3.2.3.1)，它们在高等植物中对食草动物和病原体的化学防御、木质素的生物合成和植物的生长发育具有重要作用。家族35含有β-半乳糖苷酶(EC 3.2.1.23)，在果实成熟、花衰老、碳水化合物储备动员和半乳糖脂周转等过程中起关键作用。到目前为止，只有三种拟南芥水解酶的确切生化作用是确定的。这项多学科合作研究的目的是为拟南芥基因组编码的β-葡萄糖苷酶和β-半乳糖苷酶分配生物学功能。在利用系统发育分析确定包含密切相关酶的亚家族后，将获得编码每个目标水解酶的cDNA。然后，每个水解酶将在外来宿主细胞(例如酵母和细菌)中过表达，并进行纯化，通过测量其对主要从拟南芥和相关十字花科植物中分离的一系列天然糖苷底物的活性来确定其生物学功能。在平行研究中，选定的亚家族代表的三维结构将通过同源建模和X射线衍射来确定，这将为这些水解酶如何识别和结合其底物提供新的见解。这些信息在未来改变家族1和家族35水解酶的底物专一性的研究中将是至关重要的，用于生物技术目的，包括生物量转化和改善抗食草动物防御和果实成熟。

Widely distributed in animals, plants and microbes, O-glycoside hydrolases (EC 3.2.1.-) are enzymes that catalyze the cleavage of chemical bonds between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety. Responding to the challenge of the 2010 Project to identify the function of all Arabidopsis thaliana genes within the next decade, this collaborative research will focus on approximately 75 members of two related families of glycoside hydrolases (http://www.biol.vt.edu/faculty/esen/glycosidase.lab.html and http://afmb.cnrs-mrs.fr/~pedro/CAZY/). Family 1 includes beta -glucosidases (EC 3.2.1.21) and myrosinases (EC 3.2.3.1), which function in higher plants in chemical defense against herbivores and pathogens, lignin biosynthesis, and plant growth and development. Family 35 contains the beta -galactosidases (EC 3.2.1.23), which play key roles in fruit ripening, flower senescence, mobilization of carbohydrate reserves, and galactolipid turnover. To date, the precise biochemical roles of only three of these Arabidopsis hydrolases are known with certainty. The purpose of this multidisciplinary collaborative research is to assign biological functions to beta-glucosidases and beta-galactosidases encoded by the Arabidopsis genome. After using phylogenetic analysis to identify subfamilies that contain closely related enzymes, cDNAs encoding each target hydrolase will be obtained. Each hydrolase will then be overexpressed in foreign host (e.g. yeast and bacteria) cells and purified to ascertain its biological function by measuring its enzymatic activity toward a wide range of natural glycosidic substrates isolated primarily from Arabidopsis and related crucifers for this purpose. In parallel studies, three-dimensional structures of selected subfamily representatives will be determined by homology modeling and x-ray diffraction, providing novel insights into how these hydrolases recognize and bind their substrates. This information will be of paramount importance in future research to alter the substrate specificity of Family 1 and Family 35 hydrolases for biotechnological purposes, including biomass conversion and improvements in anti-herbivore defenses and fruit ripening.