Directed divergent evolution of an enzyme active site

描述（由申请人提供）： （β/α）8（或TIM）桶提供了一个有吸引力的支架新的催化活性的定向进化。我正试图在邻琥珀酰苯甲酸合酶（OSBS）中进化分支酸酯酶（CM）活性，OSBS含有TIM桶结构域。为了实现这种活性变化，我将制作编码OSBS变体的DNA文库，其中特定的活性位点残基被改变为标准氨基酸的随机分布。为了鉴定活性CM，将对这些文库进行CM活性的体内选择。实验室进化的CM的特性可以提供对这个有趣反应的机制的洞察。为了评估TIM桶折叠对于这种定向趋异进化策略的适用性，将筛选文库的可溶性蛋白的体内产生。为了评估TIM桶折叠的酶多功能性，还将对文库进行选择以催化其他反应，例如预苯酸酯酶和酪氨酸差向异构酶活性。本研究探讨了使用一些战略性突变在TIM桶支架中创建新的催化功能的可行性。此外，本研究旨在开发改进的一般策略，以获得量身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.