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. Examination of the OSBS structure reveals a small number of residues that define the active site architecture. To redesign the OSBS active site, I will construct DNA libraries that encode variants of OSBS, with chosen active-site residues changed to a random distribution of the 20 standard amino acids. To identify active CMs, these libraries will be introduced into an in vivo selection system, selected library members may then be subjected to structural and functional analysis in vitro, as well as additional rounds of randomization and in vivo selection to further improve catalytic activity. This research examines the feasibility of using a few strategic mutations to create a completely new catalytic function in a TIM-barrel scaffold, and may shed light on mechanisms of protein evolution. The properties of a laboratory-evolved CM may also provide insight into the mechanism of this interesting reaction. Further, this study aims to develop improved strategies for obtaining tailored catalysts of organic reactions.