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

Abstract

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.

Figure 1

(a) Biocatalytic strategies of natural enzymes which catalyze aromatic alkylation/acylation; (b) organocatalytic approach to vinylogous Friedel–Crafts alkylation; (c) approach followed in this work: an artificial enzyme using expanded genetic code technology to incorporate a noncanonical catalytic residue which operates via a non-natural mechanism.

Figure 2

Analysis of the effects of mutations of residues close to the catalytic residue of LmrR_pAF in catalysis of the reaction of 1a and 2a to produce 3a. The alpha-carbon atoms of residues mutated are shown as spheres in the crystal structure of LmrR_pAF (PDB: 6I8N). Reactions were conducted under the standard reaction conditions outlined in Table 1. Enantioselectivity was visualized as the effect on relative Gibbs’ free energies of the diastereomeric transition states leading to formation of the two enantiomers of 3a which was calculated from the enantiomeric ratio according to the equation ΔΔG‡ = −RTln(e.r.). The results are shown on a scale from green (increased e.r.) to pink (decreased e.r.). Δyield values are the differences in analytical yields obtained when employing different mutants in catalysis for 3a product under the standard conditions, relative to LmrR_pAF, and are shown on a scale from blue (increased yield) to orange (decreased yield).

Figure 3

(a) Positions of residues mutated in LmrR_pAF_RGN are shown in the crystal structure of LmrR_pAF (PDB: 6I8N). (b) Analytical yields and enantioselectivities of mutants obtained throughout directed evolution of LmrR_pAF for the formation of 3a; the results with purified protein under standard conditions are outlined in Table 1. Mutations were obtained by screening of degenerate codon libraries (dashed lines) or by rational recombination (solid lines).

Figure 4

(a) Promiscuous transformations catalyzed by LmrR_pAF. (b) Directed evolution has specialized LmrR_pAF, increasing catalytic efficiency for reaction selected for, while the promiscuous activity is concomitantly diminished, giving specialized variants LmrR_pAF_RMH and LmrR_pAF_RGN.