Molecular Basis for Chemoselectivity Control in Oxidations of Internal Aryl‐Alkenes Catalyzed by Laboratory Evolved P450s

• Published in: Chembiochem : a European journal of chemical biology Vol. 25; no. 10; pp. e202400066 - n/a
• Main Authors: Soler, Jordi, Gergel, Sebastian, Hammer, Stephan C., Garcia‐Borràs, Marc
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 17.05.2024
• Subjects: Acetophenone; Alkenes; Alkenes - chemistry; Alkenes - metabolism; Biocatalysis; Biocatalysts; Carbonyl compounds; Carbonyls; Chemoselectivity; Computational Modelling; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - metabolism; Directed evolution; Enzymes; Epoxidation; Evolution; Hydrocarbons; Hydroxylation; Ketones; Oxidation; Oxidation-Reduction; P450 enzymes; Selectivity; Substitution reactions; Substrate Specificity; Substrates; Epoxidation; Computational Modelling; P450 enzymes; Hydroxylation; Chemoselectivity
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202400066

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. Computational modelling involving density functional theory (DFT) calculations, molecular dynamics (MD) simulations, and hybrid quantum mechanics / molecular mechanics (QM/MM) calculations, are used to investigate and decipher the mechanism for chemoselectivity control achieved by a set of laboratory evolved P450s for selective allylic C−H hydroxylation vs. epoxidation and carbonyl formation of internal aryl‐alkenes.