Structural and mechanistic studies of Ser/Lys dyad proteases

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The objectives of the proposed research program is to investigate the structural and mechanistic details of two unrelated families of proteolytic enzymes (one bacterial and one viral) that have evolved the same unusual catalytic mechanism. These proteases; bacterial signal peptidase and viral VP4 protease both utilize an essential lysine and serine in their hydrolytic reactions. The deprotonated lysine is thought to serve as the general-base by abstracting the proton from the serine hydroxyl thereby activating it for nucleophilic attack on the scissile carbonyl of the substrate.Signal peptidase is an essential bacterial enzyme that functions to release secreted proteins from the cell membrane and is being investigated by a number of pharmaceutical companies as a novel antibacterial target.  VP4 protease is involved in the proteolytic processing of viral coat proteins in birnavirus, the infectious agent that costs the fish farming and agricultural industry billions of dollars of lost revenue each year. We will use; molecular biology, protein chemistry, X-ray crystallography, enzyme kinetics, and Nucleur Magnetic Resonance to investigate the structural and mechanistic similarities and difference in these two enzymes families in order to provide a more detailed understanding of how the Ser/Lys dyad mechanism works, and how enzymes work in general.Canada will benefit socially and financially from the commercialized drugs that result from the structure-based rational antibacterial and antiviral drug design efforts on these enzymes.  Canada will also benefit from the training of highly qualifies personnel in our lab. Students and post-doctoral fellows are becoming structural biologist (highly sought after in both academics and industry) with skills in molecular biology, protein chemistry and X-ray crystallography.