Directed divergent evolution of an enzyme active site

描述(由申请人提供)： (β/α)8(或TIM)桶为新催化活性的定向进化提供了一个有吸引力的支架。我正在尝试进化o-琥珀酸苯甲酸合成酶(OSBS)中的分支酸变位酶(CM)活性，该酶含有TIM桶结构域。对OSBS结构的研究揭示了定义活动站点体系结构的少量残基。为了重新设计OSBS活性部位，我将构建编码OSBS变体的DNA文库，将选定的活性部位残基改变为20个标准氨基酸的随机分布。为了鉴定活性CMS，这些文库将被引入体内选择系统，然后选择的文库成员可能会在体外进行结构和功能分析，以及额外的几轮随机和体内选择，以进一步提高催化活性。这项研究考察了使用几个策略性突变在提姆-桶支架中创建全新催化功能的可行性，并可能揭示蛋白质进化的机制。实验室进化的CM的性质也可能提供对这一有趣反应的机制的洞察。此外，这项研究旨在开发获得定制的有机反应催化剂的改进策略。

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.