PUB - Publikationen an der Universität Bielefeld

P450 enzymes naturally perform selective oxidations of unfunctionalized hydrocarbon substrates, among other reactions. The adaptation of P450 enzymes to particular oxidative reactions involving alkenes is of great interest for the design of new biocatalysts. However, the mechanism that these enzymes utilize to precisely modulate the chemoselectivity and distinguishing between competing alkene double bond epoxidations and allylic C-H hydroxylations is sometimes not clear, which hampers the rational design of specific biocatalysts. In a previous work, P450LA1was engineered in the laboratory using directed evolution to catalyze the direct oxidation oftrans-b-methylstyrenetophenylacetone. The final variant, KS, was able to overcome the intrinsic preference for alkene epoxidation to directly generate a ketone productviathe formation of a highly reactive carbocation intermediate.Here, additional library screening along this evolutionary lineage permitted to serendipitously detect a mutation that overcomes epoxidation and carbonyl formation by exhibiting a large selectivity of 94% towards allylicC-H hydroxylation. A multiscalar computational methodology was applied to reveal the molecular basis towards this hydroxylation preference. Enzyme modelling suggests that introduction ofbulky substitutiondramatically changes accessible conformations of the substrate in the active site, thus modifying the enzymatic selectivity towards terminal hydroxylation and avoiding the competing epoxidation pathway. © 2024 Wiley‐VCH GmbH.