Accelerated evolution of enzyme activity to determine functional and structural tolerance

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The industrial application of enzymes is gaining in importance world-wide, as a result of their high catalytic power and the potential for modification by enzyme engineering. Our goal is to increase the scope and efficiency of enzyme engineering, by determining the tolerance of specific enzymes to intensive, targeted mutational efforts. Different enzymes appear to show different tolerance to active-site mutations. Here, we propose to study two different, unrelated enzyme systems. Following extensive modification using powerful molecular biological methodologies, we will characterize the functional variants to establish their tolerance to intensive, targeted mutations. The first system under study consists of clinically-relevant ß-lactamases which hydrolyze antibiotics such as penicillins. It is urgent to better define the molecular nature of such rapidly evolving antibiotic resistance. Artificially engineered, catalytically active chimeras of ß-lactamases will be characterized by kinetics, circular dichroism and protein NMR. These laboratory-generated ß-lactamases are an original and highly effective system for observing tolerance to high mutation load. Insight into the structure-function relationship of these artificially evolved yet functional enzymes will provide insight into the rapid development of drug resistance, while establishing a greater knowledge base for effective enzyme engineering. The second system is R67 dihydrofolate reductase, an enzyme that provides bacterial resistance to the antibiotic trimethoprim. We will identify binding scaffolds for inhibitor development to this recently evolved drug target, toward discovery of new antibiotics. In parallel, we will modify the active site of this enzyme to develop new biocatalysts for reduction of synthetically-useful prochiral imines and ketones. Comparison of the mutational tolerance and the resulting structure-function effects of these systems will contribute to advancing our capacity in enzyme engineering. Along with the development of new biocatalysts, this knowledge will serve an important industrial need.