Unlocking Iminium Catalysis in Artificial Enzymes to Create a Friedel–Crafts Alkylase

• Published in: ACS catalysis Vol. 11; no. 12; pp. 6763 - 6770
• Main Authors: Leveson-Gower, Reuben B, Zhou, Zhi, Drienovská, Ivana, Roelfes, Gerard
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 18.06.2021
• Subjects: stop-codon suppression; iminium ion; artificial enzymes; noncanonical amino acids; Friedel−Crafts alkylation; directed evolution; catalytic promiscuity
• ISSN: 2155-5435; 2155-5435
• DOI: 10.1021/acscatal.1c00996

The construction and engineering of artificial enzymes consisting of abiological catalytic moieties incorporated into protein scaffolds is a promising strategy to realize non-natural mechanisms in biocatalysis. Here, we show that incorporation of the noncanonical amino acid para-aminophenylalanine (pAF) into the nonenzymatic protein scaffold LmrR creates a proficient and stereoselective artificial enzyme (LmrR_pAF) for the vinylogous Friedel–Crafts alkylation between α,β-unsaturated aldehydes and indoles. pAF acts as a catalytic residue, activating enal substrates toward conjugate addition via the formation of intermediate iminium ion species, while the protein scaffold provides rate acceleration and stereoinduction. Improved LmrR_pAF variants were identified by low-throughput directed evolution advised by alanine-scanning to obtain a triple mutant that provided higher yields and enantioselectivities for a range of aliphatic enals and substituted indoles. Analysis of Michaelis–Menten kinetics of LmrR_pAF and evolved mutants reveals that different activities emerge via evolutionary pathways that diverge from one another and specialize catalytic reactivity. Translating this iminium-based catalytic mechanism into an enzymatic context will enable many more biocatalytic transformations inspired by organocatalysis.