Non-natural anti-Markovnikov oxidation of olefins by redirecting the oxygen transfer in P450 monooxygenases

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Developing methods to generate desired enzyme function is of high importance. This provides access to non-natural enzymes for therapeutics, biotechnology as well as organic synthesis and allows us to address challenges beyond the reach of current approaches.The present fellowship application supports a new strategy for generating non-natural enzyme function. This approach takes advantage of existing enzymes by repurposing their catalytic machinery to take on new functions. The aim of this work is the laboratory evolution of a promiscuous P450 monooxygenase for the catalytic anti-Markovnikov oxidation of olefins. While in nature P450s catalyze the epoxidation of olefins, we target to redirect oxygen transfer to generate the corresponding carbonyls from olefin oxidation. This will be performed by studying a new metalloenzyme engineering strategy, which utilizes electronic effects to tune the metal for novel catalytic cycles. Based on the significance of electronic effects in transition metal catalysis using small molecules, we expect a substantial impact in metalloenzyme design. In particular, we suppose to gain a better understanding of how electronic effects can be utilized to access non-natural enzyme activity. We will perform a detailed characterization of the evolved variants which will yield insights into the mechanistic origin of the new catalytic function.The target reaction of catalytic anti-Markovnikov oxidation of olefins is highly important in chemical synthesis, yet it has no good small-molecule catalyst solution. Since the generated enzyme function can be readily combined with other established biocatalysts, we expect further use of this novel enzyme in one-pot enzymatic cascades and in the generation of novel biosynthetic pathways. This is especially true for the new enzyme function proposed here, since the generated carbonyls are excellent functional groups for established biocatalysts (e.g., ketoreductases, transaminases, aldolases, ThDP-dependent lyases, hydroxynitrile lyases and Pictet-Spenglerases). Therefore, several anti-Markovnikov functionalizations such as formal anti-Markovnikov hydration and hydroamination could be realized with enzyme cascades, which is a highly attractive application of this work.