KSEF: R&D Excellence: Engineering Desired Catalytic Activity into Superoxide Dismutase

We propose to re-engineer the active site of superoxide dismutase (SOD) to perform 2-oxo acid-dependent dioxygenation. Enzymes are catalysts extraordinaire, offering highly specific and efficient chemical conversions under benign conditions. Thus they are an economical yet extremely powerful enabling technology. 2-oxo-acid-dependent dioxygenases (20AdOs) catalize diverse reactions involved in antibiotic synthesis, waste detoxification, plant hormone production and human metabolism. These reactions are exceptionally difficult to perform synthetically, making the 20AdOs strategic targets for enzyme engineering. We have extensive experience understanding and manipulating the catalytic activity of SOD, which is a close cousin to the 20AdOs but is exceptionally well-behaved and well understood. To support 20AdO chemistry, we have designed and generated well-behaved SOD mutants with expanded substrate binding cavities. We now propose to evaluate these mutants' abilities to bind a range of 2-oxo acid substrates and identify substrates which undergo the desired oxidative decarboxylation when bound to the engineered enzyme. A set of second generation substrates incorporating an aromatic functionality to be oxidized, and mimicking herbicide and phenolic pollutants, will be the targets of two second-generation engineered enzymes. Spectroscopic and structural studies will address the mode of substrate binding, and identify spectroscopic signatures of activity. Thus we will identify 20As that support novel reactivity in engineered SOD, as well as amino acids which should be mutated to expand and modify SOD's reactivity further. By combining mutagenesis with systematic characterization of the substrate specificity and reactivity that results, we also guarantee accrual of the knowledge base necessary to refine our engineering strategy and improve the reactivity we obtain. This will set a precedent for re-engineering metalloenzymes.