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。为了实现这种活性的变化，我将建立编码OSBS变体的DNA文库，将特定的活性位点残基改变为标准氨基酸的随机分布。为了识别活性CMS，这些文库将受到CM活性的体内选择。实验室进化的CM的性质可能提供对这一有趣反应的机制的洞察。为了评估Tim桶折叠是否适合这一定向分化进化策略，将对文库进行筛选，以用于体内生产可溶性蛋白。为了评估TIM桶折叠的酶的多功能性，文库还将被用于其他反应的催化，如预苯酸脱水酶和酪氨酸异构体酶的活性。这项研究考察了在提姆-桶支架中使用几个有策略的突变来创建新的催化功能的可行性。此外，这项研究的目的是开发改进的通用策略，以获得定制的有机反应催化剂。

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.