InDel mutagenesis in active site loops to engineer enzymes

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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.