(Focused) Directed Evolution to Unterstand Relationships in alpha/beta-Hydrolase-Fold Enzymes

酯酶、环氧化物水解酶和卤代醇脱卤酶具有高度保守的蛋白质骨架((/(-)水解酶折叠)，关键氨基酸残基的差异很小。然而，改变保守的氨基酸(即酯酶中的丝氨酸为天冬氨酸以产生环氧化物水解酶活性)不会导致功能变化。在这项提案中，合理的蛋白质设计和定向进化的组合将被应用于创造来自荧光假单胞菌(PFE)的酯酶的催化混杂活性。初步实验已经证实，该方法可以获得环氧化物水解酶活性。这将通过进一步的定向进化、反向突变和饱和突变来延长。我们希望对授予混杂活动所需的机制和关键残留物有一个详细的了解。这种对结构-功能基础的洞察不仅应该扩大我们对环氧化物水解酶如何工作的理解，而且还可以提供关于a/？-水解酶功能的一般信息。同样的概念将被应用于授予PFE中的卤醇脱卤酶活性。

Esterases, epoxide hydrolases and haloalcohol dehalogenases have a highly conserved protein scaffold ((/(-hydrolase fold) with (apparently) only minor differences in key amino acid residues. However, changing conserved amino acids (i.e. the serine in the esterase to an aspartate to create epoxide hydrolase activity) does not lead to functional changes. In this proposal, a combination between rational protein design and directed evolution will be applied to create catalytically promiscuous activity into the esterase from Pseudomonas fluorescens (PFE). Preliminary experiments already confirmed that epoxide hydrolase activity could be gained by this approach. This will be extended by further rounds of directed evolution, back mutations and saturation mutagenesis. We expect to get a detailed picture of the mechanism and key residues required to confer the promiscous activity. This insight into the structure-function basis should not only extend our understanding how epoxide hydrolases work, but also provide information about a/ß-hydrolase function in general. The same concept will be applied to confer haloalcohol dehalogenase activity in PFE.