Molecular Structural Studies of Proteins and Enzymes Important to Biotechnology

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My research program focuses on developing a molecular structural framework for understanding and manipulating proteins important for biotechnology. The discoveries made from my research program have helped and will help to attract investment and train HQP for biotechnology in Canada. The three main projects in this proposal follow a unified structural biology approach to make advances on systems at the leading edge of biotechnology worldwide. The first project aims to engineer novel forms of streptavidin for reversible coupling applications. I propose to use our recently determined structure of streptavidin bound to the SBP-Tag to design a new generation of commercially attractive affinity-based reagents. Disulfide-linked variants and other designs are being expressed and characterized. The designs that yield the best coupling efficiencies and kinetics will be structurally characterized to help improve binding affinity and specificity. The second project will reveal novel structure-function relationships in plant phosphoprotein phosphatases (PPP). Preliminary work on AtRLPH2 has yielded well-diffracting crystals (dmin=2.0Å). Structure determination will use selenomethionine or tungstate derivatives. Novel structures of complexes with peptide ligands and targeting subunits will also provide a basis for understanding the mechanisms underlying specificity, catalysis and targeting. The third project establishes a molecular structural framework for understanding key enzymes responsible for the biosynthesis of benzylisoquinoline alkaloids (BIA), which include the commonly used analgesics codeine and morphine, as well as many antimicrobial, antitussive and anticancer compounds. Our initial work has yielded well-diffracting crystals of pavine N-methyltransferase (PavNMT) from Thalictrum flavum, and we have recently solved and refined the structure of a binary complex with the methyl donor S-adenosylmethionine (SAM) to 2.0 Å resolution. We are currently crystallizing and solving the structures of two ternary complexes (SAM and the substrate-analogue inhibitor tetrahydropapaverine, or S-adenosylhomocysteine and the substrate pavine). The initial structural information on PavNMT suggests mechanisms for methyl transfer and substrate specificity that we will further explore using site-directed mutagenesis, enzyme kinetics, crystallography and computational methods. We have also initiated structural studies on other key enzymes important for BIA biosynthesis. These studies promise to reveal new insights into substrate recognition and catalysis for structurally uncharacterized classes of enzymes essential to BIA biosynthesis. This information will provide critical tools for improving metabolic engineering initiatives aimed at generating modified BIA compounds with novel pharmacological properties.