Origins of Enantioselectivity in Enzymatic Oxidation of Indoles
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Authors
Amoako, George
Narayan, Alison
Issue Date
2024-04-04
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Abstract
Oxidized indole motifs are observed across disciplines in natural products, dyes, and electronic materials. Oxidizing indoles with current organic oxidants often give rise to complex mixtures of oxidation products and racemic mixtures. An enzymatic method to oxidize 2,3-disubstituted indoles to the tertiary alcohol has been developed in the Narayan lab, forming 3- hydroxyindolenines in up to 95:5 enantiomeric ratio. Despite exhibiting excellent enantioselectivity for some substrates, others are racemic. It’s not understood why this enzyme produces an enantioenriched product from an achiral starting material in some cases. Using substrate docking, six amino acid residues potentially playing significant roles in substrate binding and enzyme mechanism via hydrogen bonding and π-stacking have been identified and mutated with site-directed mutagenesis. The mutated enzyme has been expressed in E. coli and we performed enzymatic reactions to observe changes in reaction yield and enantioselectivity. Binding mode in the enzyme affects enantioselectivity as the Y245L mutant resulted in a flip in enantioselectivity such that the mutant enzyme produces the majority of the least favored enantioselective product in the wild type. Removing the Tyr245 π-π interaction significantly affects the enantioselectivity of the enzyme as it loses the ability to effectively bind to the substrate. Removing the D54L hydrogen bonding interaction also affects enantioselectivity. This shows that the enzyme’s effective oxidation of indole motifs is due to its ability to sufficiently bind to the substrates in the right orientation and with the proper interactions.