InDel mutagenesis in active site loops to engineer enzymes

酶工程领域发展成为一项成熟的技术，影响着工业生物催化、生物医学和合成生物学等多个领域。与定向进化、合理设计甚至计算从头设计等酶工程策略无关，蛋白质工程师主要依靠氨基酸取代来优化蛋白质。这与自然方法形成了鲜明的对比，在自然方法中，除了氨基酸取代外，包括插入和缺失(Indels)在内的骨架修饰是关键因素。虽然一些单独的研究成功地应用氨基酸吲哚来改良酶，但我们缺乏对这种方法的系统探索。考虑到INDELS在自然进化中的成功，不难相信INDELL工程学有潜力成为一项跨越式技术。我建议系统地探索Indel主动站点环路的工程。我们把Rieske非血红素双加氧酶作为一类新兴的生物催化剂来研究，因为我们在它们的活性位点环中发现了特别重要的Indels。这项拟议的研究将通过使用不同的策略整合indels，并通过解决酶工程中的三个不同挑战，即产品选择性、底物范围和获得非生理化学的途径，来验证Indel工程。所有的目标反应都具有很高的合成价值和/或具有环境意义。我们的目标是解释产生的indels对酶结构和功能的影响。

The field of enzyme engineering developed to a mature technology that impacts various areas such as industrial biocatalysis, biomedicine and synthetic biology. Independent of the enzyme engineering strategy, such as directed evolution, rational design or even computational de novo enzyme design, protein engineers mainly rely on amino acid substitutions to optimize proteins. This is in stark contrast to natures approach, where next to amino acid substitutions, backbone modifications including insertions and deletions (InDels) are key players. While a few individual studies successfully applied amino acid InDels to improve enzymes, we lack a systematic exploration of this approach. Given the success of InDels in natural evolution, it is not difficult to believe that InDel engineering has the potential to become a leapfrog technology. I propose to systematically explore InDel engineering of active site loops. We focus on Rieske non-heme dioxygenases as emerging class of biocatalysts since we found particularly significant InDels in their active site loops. The proposed research will validate InDel engineering by using different strategies to incorporate InDels and by addressing three different challenges in enzyme engineering, namely product selectivity, substrate scope and access to non-physiological chemistry. All target reactions are of high synthetic value and/or of environmental significance. We aim to explain the consequences of the generated InDels for enzyme structure and function.