Characterizing the link between protein dynamics and catalytic function to improve the design of enzyme biocatalysts

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Enzymes are increasingly being used in industrial and pharmaceutical settings, primarily as a cost effective, environmentally friendly alternative to harmful organic solvents. Yet, the engineering of new enzymes dedicated to specific applications remains a very arduous and time consuming endeavour that often yields inefficient biocatalysts. This is mainly attributed to a lack of understanding of how enzyme engineering affects the 3D structure, catalytic function, and molecular flexibility of enzymes. Recent experimental evidence indicates that several concerted molecular motions occurring on the timescale of the catalytic reaction play an important role in promoting catalysis in numerous enzyme systems. However, we have yet to understand how this atomic flexibility couples to the catalytic event, and whether enzymes with similar structures and/or function also retain comparable molecular motions. Moreover, the effect of the amino acid sequence on the transmission of this dynamic molecular signal remains unknown. To address these critical enzyme engineering issues, our work will focus on members of the ribonuclease superfamily, which includes an extensive network of distinct biocatalysts involved in bactericidal, angiogenic, cytotoxic and anti-tumoural activities. Using an innovative combination of molecular biological techniques and nuclear magnetic resonance (NMR), we will study the effect of single and combinatorial mutations on the molecular flexibility and catalytic function of ribonucleases. By providing clues relating to the modulation of catalytic activity by controlling molecular motions using mutagenesis, our research program has the potential to lead to fundamental breakthroughs in the field of enzyme engineering applied to biocatalysts of industrial and pharmaceutical relevance. Additionally, by characterizing molecular motions that contribute to catalysis in several ribonucleases with critical cellular function, the proposed research will provide valuable information on the potential allosteric modulation of these clinically relevant targets.