Reaching enzymatic perfection of the de novo designed metalloprotein MID1sc10 and implementing of new enzymatic activities into a flexible protein scaffold by exchanging metal ions and directed evolution

从头设计的金属蛋白 MID1sc10 达到酶促完美，并通过交换金属离子和定向进化将新的酶促活性实施到柔性蛋白支架中

• 批准号: 430981304
• 负责人: Dr. Dominic Hoch
• 金额: --
• 依托单位: Laboratorium für Organische Chemie
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/430981304?language=en
• 关键词: Reaching; enzymatic; perfection; de; novo

This proposal aims at improving the esterase activity of the metalloprotein MID1sc10 to reach enzymatic perfection as well as introducing novel enzymatic activities into this de novo designed peptide scaffold applying rational design and laboratory evolution. The artificial peptide scaffold MID1sc that will be used in this study consists of two helix-turn-helix motifs, a zinc coordination site and a hydrophobic pocket. Because of the high plasticity of the scaffold, it was possible to evolve it to a highly active metalloprotein with esterase activity. The initial MID1 also showed a slight phosphatase activity, a perfect starting point to evolve this structure to a highly active enzyme. Moreover, using rational design, we want to introduce amidase activity as a novel enzymatic functionality into the scaffold. In a first step, we plan to exchange the complexed metal ion in the MID1 scaffold and evaluate the amidase and phosphatase activity. As subsequent steps, laboratory evolution applying focused and random mutagenesis will be used to further enhance the activities and ultimately yield an efficient enzyme. For both proposed projects, the use of an established fluorescence-based microfluidics setup in the Hilvert lab that allows high-throughput screening of large DNA libraries. In order to understand the underlying mechanisms of enzyme catalysis and evolution, any successfully evolved enzyme variant will be characterized using X-ray crystallography as well as enzyme kinetics measurements.

该建议旨在改善金属蛋白MID1SC10的酯酶活性，以达到酶促的完美，并将新型酶促活性引入该从头设计的肽支架中，并采用合理的设计和实验室进化。本研究中将使用的人造肽支架MID1SC包括两个螺旋 - 旋转螺旋序，一个锌协调位点和一个疏水口袋。由于支架的可塑性很高，因此可以将其进化为具有酯酶活性的高活性金属蛋白。最初的MID1还显示出轻微的磷酸酶活性，这是将这种结构演变为高度活性酶的理想起点。此外，使用有理设计，我们希望将amidase活性作为一种新型酶促功能引入脚手架。第一步，我们计划在MID1支架中交换复合的金属离子，并评估酰胺酶和磷酸酶活性。随后，将使用专注和随机诱变的实验室进化来进一步增强活性并最终产生有效的酶。对于两个提出的项目，在Hilvert Lab中使用已建立的基于荧光的微流体设置，该设置允许对大型DNA文库进行高通量筛选。为了理解酶催化和进化的潜在机制，使用X射线晶体学以及酶动力学测量结果来表征任何成功进化的酶变体。