An artificial metalloenzyme with the kinetics of native enzymes

Abstract

Natural enzymes contain highly evolved active sites that lead to fast rates and high selectivities. Although artificial metalloenzymes have been developed that catalyze abiological transformations with high stereoselectivity, the activities of these artificial enzymes are much lower than those of natural enzymes. Here, we report a reconstituted artificial metalloenzyme containing an iridium porphyrin that exhibits kinetic parameters similar to those of natural enzymes. In particular, variants of the P450 enzyme CYP119 containing iridium in place of iron catalyze insertions of carbenes into C-H bonds with up to 98% enantiomeric excess, 35,000 turnovers, and 2550 hours^-1 turnover frequency. This activity leads to intramolecular carbene insertions into unactivated C-H bonds and intermolecular carbene insertions into C-H bonds. These results lift the restrictions on merging chemical catalysis and biocatalysis to create highly active, productive, and selective metalloenzymes for abiological reactions.

Fig. 1.. Structure of WT Fe-CYP119.

Image was prepared in Chimera from Protein Data Bank 1IO7. (Left) Complete structure of Fe-CYP119. (Right) Active-site residues modified during directed evolution of the protein scaffold to increase activity and selectivity for carbene insertions into C–H bonds.

Fig. 2.. Directed evolution of Ir(Me)-PIX CYP119 for enantioselective insertions of carbenes into C–H bonds.

(A) Model reaction converting diazoester 1 to dihydrobenzofuran 2. (B) Enantioselectivity and yields for the formation of 2 catalyzed by evolved variants of CYP119 (0.17% catalyst loading, 10 mM substrate). (C) Kinetic parameters describing the formation of 2 by variants of CYP119 (0.1 mol % catalyst loading, 5 mM substrate). For free Ir(Me)-PIX, k₁ (the first-order kinetic constant) is listed instead of k_cat/K_M (16). (Inset) Dependence of TOF on the initial concentration of 1 for reactions using 0.005 mM catalyst. (D and E) Comparison of K_M and k_cat values for CYP119-Max with those of natural enzymes involved in the metabolism of intermediate and secondary metabolites (5); for comparison, the kinetic parameters for Ir(Me)-PIX mOCR-myoglobin (H93A and H64V) catalyzing the same transformation are shown.

Fig. 3.. Selective variants of Ir(Me)-PIX CYP119.

These variants catalyze enantioselective intra- and intermolecular C–H carbene insertion reactions of activated and unactivated C–H bonds. (A to D) Intramolecular C–H carbene insertion reactions. Reactions were conducted at room temperature unless otherwise noted. (E) An intermolecular C–H carbene insertion reaction. Conditions: 10 (10 μmol) and EDA (100 μmol). EDA was added as a 50% solution in N,N′-dimethylformamide over 1 hour by use of a syringe pump.

Fig. 4.. Productivity of Ir(Me)-PIX CYP119.

Intramolecular C–H carbene insertion reactions of substrate 1 catalyzed by Ir(Me)-PIX CYP119-Max under synthetically relevant reaction conditions.