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oai:recercat.cat:10256/250832024-10-29T23:15:21Zcom_2072_452955com_2072_2054col_2072_453073

00925njm 22002777a 4500 dc Soler i Parpal, Jordi author Gergel, Sebastian author Hammer, Stephan C. author Garcia Borràs, Marc author 2024-05-17 P450 enzymes naturally perform selective hydroxylations and epoxidations of unfunctionalized hydrocarbon substrates, among other reactions. The adaptation of P450 enzymes to a particular oxidative reaction involving alkenes is of great interest for the design of new synthetically useful 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, a P450 from Labrenzia aggregata (P450LA1) was engineered in the laboratory using directed evolution to catalyze the direct oxidation of trans-β-methylstyrene to phenylacetone. The final variant, KS, was able to overcome the intrinsic preference for alkene epoxidation to directly generate a ketone product via the 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 allylic C−H hydroxylation. A multiscalar computational methodology was applied to reveal the molecular basis towards this hydroxylation preference. Enzyme modelling suggests that introduction of a bulky substitution dramatically changes the accessible conformations of the substrate in the active site, thus modifying the enzymatic selectivity towards terminal hydroxylation and avoiding the competing epoxidation pathway, which is sterically hindered Research Funding: Generalitat de Catalunya AGAUR Beatriu de Pinós. Grant Number: H2020 MSCA-Cofund 2018-BP-00204; 2021-SGR-00623; Spanish MICINN (Ministerio de Ciencia e Innovación). Grant Numbers: PID2019-111300GA−I00, PID2022-141676NB−I00, TED2021-130173B−C42; Ramón y Cajal. Grant Number: RYC 2020–028628-I fellowship; Deutsche Forschungsgemeinschaft. Grant Number: 420112577 and Spanish MIU (Ministerio de Universidades). Grant Number: FPU18/02380 Open Access funding provided thanks to the CRUE-CSIC agreement with Wiley http://hdl.handle.net/10256/25083 Enzims Enzymes Grups funcionals Functional groups Reaccions químiques Chemical reactions Molecular Basis for Chemoselectivity Control in Oxidations of Internal Aryl-Alkenes Catalyzed by Laboratory Evolved P450s