CAREER: Glycoside Hydrolase Processivity and Substrate Recognition Mechanisms

Overview The PI’s long-term career goal is to define general, fundamental relationships between glycoside hydrolase (GH) structure and overall performance and stability. Accordingly, the research goal of this career development plan is to establish a molecular-level understanding of the mechanisms governing GH processivity and substrate binding. Three objectives are proposed, integrating computationally-driven discovery with experimental characterization of enzyme reactions and ligand binding. (1) The Serratia marcescens chitinase model system will be used to uncover dynamic and thermodynamic contributions of individual residues to substrate binding and processive ability. Molecular dynamics simulations and free energy calculations of chitinase variants, alongside determinations of processive ability, will be used to define the effect of active site perturbations on processivity. (2) A hypothesized mathematical relationship between ligand binding free energy and processivity will be tested using free energy calculations and isothermal titration calorimetry coupled with determination of kinetic parameters in chitinases. This relationship is expected to be general to all GHs, given that chemical composition and structure of the active site defines ligand binding. (3) The transferability of findings from the chitinase system to an industrial cellulase will be examined. A computationally selected set of variants and subsequent characterization will determine if description of processive ability is general across GHs. Empirical models within families will be considered as an alternative approach. The educational objective of this proposal is to increase access to and understanding of biocatalysis across generations of developing scientists and engineers and among underserved demographics including rural populations and young women. Toward this objective, three activities are proposed. (1) Mentored international research experiences will be incorporated into graduate education, where funded students spend summers physically located at collaborators’ labs in Norway, Sweden, and Estonia. (2) In response to a dearth of high performance computing educational resources, the PI and her undergraduate researchers will develop online training modules designed to reach the nation’s rural and underrepresented scientists. (3) The PI will expand her K-12 outreach by developing a new biocatalysis demonstration for local girls STEM events.