ANALYSIS OF THE MODULAR ARCHITECTURE OF TWO BLOOD GROUP ACTIVE GLYCOSIDE HYDROLA

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 肺炎链球菌是人类胃肠道的一种重要的定殖菌。其分泌的毒力因子的主要成分是碳水化合物活性酶，它是一种大的、多模块的酶，参与从宿主组织中获取和加工多糖。我们选择了两个对人类血型抗原有活性的98家族糖苷水解酶进行鉴定。我们已经用X射线结晶解决了所有单个模块的高分辨结构；然而，全长酶的结晶被证明是棘手的。为了构建一个经过实验验证的整个酶的工作模型，基于这些酶的单个模块结构的3D结构，我们正在SSRL的束线4-2上申请SAXS实验。该方法提供了一种独立确定模块的相对方向的方法，并帮助基于该方法确定的低分辨率解结构建立模型。正如最近在几个案例中成功地表明的那样，全长酶的结构研究可以通过溶液中的SAXS测量来获得。这让我们看到了一些关于催化和粘附性的有趣方面。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Streptococcus pneumoniae is a notable colonizer of the human gastrointestinal tract. A major component of its secreted virulence factors are carbohydrate active enzymes which are large, multi-modular, and involved in harvesting and processing glycans from host tissues. We have chosen two family 98 glycoside hydrolases for characterization which are active on human blood group antigens. We have solved the high resolution structures of all the individual modules using x-ray crystallization; however, crystallization of the full length enzyme has proven intractable. In order to construct an experimentally proven working model of the whole enzyme, based on the 3D structures of the individual modular constructs from these enzymes, we are applying for SAXS experiments at the SSRL on beam line 4-2. This method provides a way of independently determining the relative orientations of the modules and helping to build a model based on the low resolution solution structure determined by this method. As has been shown successfully in several cases recently, the structural study of full-length enzymes can be attained by SAXS measurements in solution. This allows us to see some interesting aspects regarding catalysis and adherence.