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

Stanford University, Stanford CA

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Abstract

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.