Grant: Discovery Grants Program - Individual

My research program uses a molecular structural approach to understand and engineer key enzymes and binding proteins important for biotechnology, especially metabolic engineering. Project 1 aims to engineer novel forms of streptavidin with uniquely useful redox-switchable binding properties. We recently made a breakthrough, discovering how the introduction of disulphide bonds into a key loop in streptavidin creates redox-dependent binding to biotin. Breaking one of these disulphide bonds with a mild reducing agent increases the rate of dissociation for biotin by over a thousand-fold, allowing for the first time the reversible formation and breakdown of tight-binding biotin complexes under physiological conditions. This mutein is useful for certain applications but binds too tightly for applications where fast dissociation kinetics are required. To address this shortcoming, we propose a structure-based strategy to design and characterize a suite of next-generation muteins with faster dissociation kinetics in the reduced state while retaining extremely slow dissociation kinetics in the oxidized state. These muteins would have many applications in biotechnology, including immediate applications to discover protein interaction partners in projects 2 and 3 of this proposal. Project 2 explores structure-function relationships in novel members of the phosphoprotein phosphatase (PPP) family in plants. By solving the structure and characterizing the unusual substrate specificity of AtRLPH2, we recently discovered a novel mechanism to recognize dually phosphorylated substrates, corroborating phosphoproteomic data implicating RLPH2 in the regulation of MAP kinase cascades. Using tools from project 1, I propose to further explore how AtRLPH2 regulates MAP kinases, as well as to determine foundational structure-function relationships for other noncanonical plant protein phosphatases, such as AtSLP1. Project 3 further develops studies on enzymes central to the biosynthesis of benzylisoquinoline alkaloid (BIA) compounds. Our foundational structure-function studies of N-methyltransferasse (NMTs) have established key molecular mechanisms underlying specificity and catalysis. These findings indicate how to design NMT muteins with novel specificities, creating new tools for metabolic engineering. We have also undertaken structure-function studies of BIA O-methyltransferases, as well as the enzymes in the terminal steps of the pathway converting thebaine to codeine and morphine. Our recent structure-function studies of codeinone reductase suggest the possible importance of novel protein-protein interactions with wider implications on the biosynthesis of secondary metabolites. Tools from project 1 will help us explore some of these protein-protein interactions. My research program focuses on problems that will have both short-term applications in Canada's rapidly growing biotechnology industry, as well as long-term applications advancing basic knowledge in biology.