Exploration of the regulation and structure-function relationships of enzymes and proteins involved in sulfur amino acid metabolism and storage in bacterial and plant systems

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Protein engineering and other nanoscale sciences possess remarkable potential to provide new environmentally friendly technologies to address a number of the challenges facing society. My lab is well positioned for the transdisciplinary approach required for this research. Our distinct blend of expertise spans protein biochemistry, microbiology and plant science. The resulting opportunities for knowledge translation (e.g. between biochemistry and plant science) and the diversity of techniques employed provide a strong training environment. The primary focus of my research program is amino acid metabolism and we have focused on protein structure-function relationships. The ubiquitous nature of these themes provides the basis for our studies in diverse systems, as illustrated by the two ongoing projects described in this proposal: 1. Engineering pyridoxal 5’-phosphate enzymes. Proteins are large biological molecules, formed of individual building blocks called amino acids, which fold in a complex three-dimensional pattern. This enables the unique and specific function of each of the thousands of distinct proteins inside a living cell. One of the many roles of proteins is in the catalysis of chemical reactions. Enzymes are large biological molecules, generally proteins, which catalyze biochemical reactions, converting the starting `substrate` to a specific `product` under the physiological conditions of the living cell. The economic prospects of tailoring enzymes for biotechnology have spurred investments and interest in protein engineering research. The 2010 global market value of industrial enzymes was $3.3 US billion, a value predicted to grow steadily. Enzymes catalyzing transformations of amino acids are generally dependent on pyridoxal 5’-phosphate (PLP). The versatility of the PLP cofactor is such that it can catalyze an array of reactions. We will continue our research probing the complex and subtle structure-function relationships that underlie specificity in these enzymes. Our long-term goal is the application of this knowledge to modify their specificity to meet the requirements of industrial processes. 2. Modifying the amino acid balance of plants to improve their nutritional properties. Grain legumes, such as chickpea, provide approximately 10% of the world supply of dietary protein and produce approximately $2.7 billion (CDN) in export revenue for Canada, annually. However, the seeds are deficient in the essential amino acid methionine, containing on average 5-fold less than the recommended 3-5% recommended for human nutrition. Reported attempts to modify the amino acid content of the harvested portion of a given crop plant can be classified as either ‘push’ or ‘pull’ strategies. Push approaches alter the flux of metabolites in the biosynthetic pathway of the deficient amino acid, thereby yielding an increase in the soluble amino acid pool. The complementary pull strategy focuses on the endpoint, attempting to increase the content of the target amino acid in the storage proteins of the seed. We are working to: (1) develop a thorough understanding of the regulation of these biosynthetic enzymes, in the context of the target plant species, in order to more subtly increase the soluble methionine pool and (2) establish a two-phase screen to enable engineering of the native seed storage proteins to modify their amino acid profile. This strategy is novel and provides an example of knowledge translation between scientific disciplines. Our research will provide the knowledge base required to improve the nutritional quality of grain legumes. Our innovative two-phase screen also has the potential to be applied to engineer the amino acid profiles of other crops (e.g. increasing the lysine content of cereals).