Grant: Discovery Grants Program - Individual

The enzymes involved in the breakdown of complex sugars (glycosidases) play a number of important biological roles, ranging from the degradation of polysaccharides such as starch and cellulose in digestive processes to the exquisitely selective trimming' of glycoproteins required for individual cells to interact with each other and with their environment. A detailed understanding of how these highly efficient catalysts work is valuable, not only for understanding catalysis itself but also in the engineering of these enzymes to carry out novel reactions. It also enables the design and synthesis of specific inhibitors to control cellular processes, thus to possibly act as therapeutics. This proposal comprises three parts. In the first, we will develop new high-throughput approaches to discover novel glycosidases derived from vast populations of microorganisms that have never been studied. This will be accomplished by extracting whole DNA from environments of interest, such as forest soils and placing large sections of that DNA into a standard laboratory bacterium such as E. coli. We shall use nanodroplets of water in oil as tiny reaction vessels for each individual bacterium, and develop detection systems that allow us to identify which droplets contain E. coli that are producing the enzymes we want by expressing environmental DNA genes. We shall use this screening approach to find a range of new enzymes, including those that can be used for cutting whole sugar glycans from the surface of proteins and cells. These will be valuable in the analysis of glycoproteins and in modifying cell surfaces. In the second part, we shall follow up on our recent demonstration that unnatural “pseudo sugars” are recognized and hydrolyzed by glycosidases. However, this occurs very slowly, despite their inherently similar reactivity to regular sugars. We shall “retrain” glycosidases to hydrolyze these pseudosugars just as fast as they do normal sugars through the process of “directed evolution”. We will then further reprogram them to attach these pseudosugars onto other sugars and cell surfaces, thereby generating novel, metabolically stable sugar mimics that may be useful as materials or ultimately in therapeutics. Finally we shall decipher the enzymatic mechanism by which a key sugar involved in plant cell walls, arabinofuranose, is synthesized. The insights gained may be useful in the design of herbicides and anti-bacterials. Overall our work will provide fundamental insights into the biochemistry of sugars and enzymes, as well as providing novel approaches for discovery of industrially and medically useful enzymes. This research program will also provide excellent training for new scientists.