Investigation of phenylalanine ammonia lyase and processing alpha glucosidase I

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Enzymes are highly evolved proteins, exquisitely designed by nature for specific purposes. By fully understanding their mechanism of action and structure, we can exploit them as efficient biosynthetic tools, modulate biochemical pathways to obtain desired end-products, or develop inhibitor that can control activity-associated disease states. Two different enzymes from two yeasts will be investigated. Processing glucosidase I (Glu I) is critical to cell function in higher eukaryotes and has potential as a therapeutic target for anti-viral , anti-inflammatory, and anti-cancer drugs. Tyrosine/phenylalanine ammonia lyases (T/PAL) are enzymes with a wide range of specificty towards phenylalanine or tyrosine. The basis for this specificity is not understood. Activity with tyrosine is highly desirable, as the product, p-coumaric acid, has a high industrial demand. A better understanding of the mechanism and structure of Glu I and T/PAL is necessary to control or use these enzymes in health and commercial applications. In terms of basic research, both of these enzymes pose intriguing questions related to enzyme structure, function, and mechanism. This project will carry out the following: (1) site directed mutagenesis of potential key amino acid residues involved in substrate binding and catalysis , followed by kinetic and structural evaluation of the mutants; (2) examining the functional domain structure of the enzymes by cloning and expression of truncated gene products; (3) crystallographic studies to further our understanding of the enzyme structure/function. These experiments will clarify key aspects of the structure and mechanism of Glu I and T/PAL, and will allow us to exploit them through rational inhibitor synthesis (Glu I) and by designing enzymes to efficiently produce industrially important p-coumaric acid (T/PAL).