Structure-function studies of chitinases and cellulases - the molecular mechanism of enzymatic conversion of crystalline polysaccharides

Despite years of research there are still major holes in our understanding of how enzymatic machineries acting on recalcitrant polysccharides such as cellulose and chitin accomplish their degradative tasks. Chitin and cellulose are structurally similar an d cellulases face the same challenges during cellulose degradation as chitinases face during chitin degradation. There is a big need for fundamental research on these enzymes. In this project, will explore two aspects of these enzymatic machineries that a re gaining increased attention partly because of recent findings in our own research group. By carrying out structure-function studies of chitinases and cellulases, we will study how enzymes accomplish processivity and how processivity contributes to enzy me efficiency. Furthermore, we will study how carbohydrate-binding domains of modular cellulases and chitinases contribute to processivity and to enzyme efficiency in general. Finally, recent work from our group and others shows that organisms may produce independent accessory helper proteins that improve enzyme efficiency by increasing substrate accessibility. We will study the interplay between such accessory proteins and processive or non-processive chitinase and cellulase variants. Most importantly, t hese "classical" structure-function studies will be complemented by the use of single-molecule techniques for creating novel insight in how wild-type and mutant enzymes and domains bind to, affect and process single-chained and crystalline polysaccharide substrates. These analyses, to be carried out in collaboration with expert laboratories, include the use of optical tweezers, total internal reflection fluorescence microscopy (TIRF), dynamic force microscopy (DFM) and atomic force microscopy (AFM).