Grants and Contracts Details
Description
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.
Status | Finished |
---|---|
Effective start/end date | 4/1/02 → 3/31/04 |
Funding
- KY Science and Technology Co Inc: $95,000.00
Fingerprint
Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.