Directed divergent evolution of an enzyme active site

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DESCRIPTION (provided by applicant): The (beta/alpha)8 (or,TIM) barrel provides an attractive scaffold for the directed evolution of new catalytic activities. I am attempting to evolve chorismate mutase (CM) activity in o-succinylbenzoate synthase (OSBS), which contains a TIM barrel domain. To effect this change in activity, I will make DNA libraries that encode OSBS variants, with specific active site residues changed to a random distribution of standard amino acids. To identify active CMs, these libraries will be subjected to in vivo selections for CM activity. The properties of a laboratory-evolved CM may provide insight into the mechanism of this interesting reaction. To assess the suitability of the TIM barrel fold for this strategy of directed divergent evolution, the libraries will be screened for the in vivo production of soluble protein. To assess the enzymatic versatility of the TIM barrel fold, the libraries will also be subjected to selections for catalysis of other reactions, such as prephenate dehydratase and tyrosine epimerase activities. This research examines the feasibility of using a few strategically placed mutations to create a new catalytic function in a TIM-barrel scaffold. Further, this study aims to develop improved general strategies for obtaining tailor-made catalysts of organic reactions.