YfeX - A New Platform for Carbene Transferase Development with High Intrinsic Reactivity

Abstract

Carbene transfer biocatalysis has evolved from basic science to an area with vast potential for the development of new industrial processes. In this study, we show that YfeX, naturally a peroxidase, has great potential for the development of new carbene transferases, due to its high intrinsic reactivity, especially for the N-H insertion reaction of aromatic and aliphatic primary and secondary amines. YfeX shows high stability against organic solvents (methanol and DMSO), greatly improving turnover of hydrophobic substrates. Interestingly, in styrene cyclopropanation, WT YfeX naturally shows high enantioselectivity, generating the trans product with 87 % selectivity for the (R,R) enantiomer. WT YfeX also catalyzes the Si-H insertion efficiently. Steric effects in the active site were further explored using the R232A variant. Quantum Mechanics/Molecular Mechanics (QM/MM) calculations reveal details on the mechanism of Si-H insertion. YfeX, and potentially other peroxidases, are exciting new targets for the development of improved carbene transferases.

Keywords: N−H insertion; QM/MM calculations; biocatalysis; carbene transfer; cyclopropanation.

Scheme 1

Cyclopropanation mechanism of heme enzymes via carbene insertion.[ ⁷ , ⁸ ]

Figure 1

Pymol representation of the crystal structure of YfeX (left), and of the active site with important SCS amino acids highlighted (PDB code: 2IIZ).

Figure 2

Time course of the N−H insertion reaction of aniline catalyzed by YfeX. The catalyst was prepared at 20 μM concentration and reduced with 500 equivalents of Na₂S₂O₄ before addition of 1000 equivalents of aniline and 2000 equivalents of EDA to a final volume of 500 μL. The organic products were extracted with 3 mL of ethyl acetate after the following time points: 2, 5, 10, 30, and 60 minutes. Finally, the product was quantified by GC/MS. Exponential fit of the data gives a k_obs=0.210 min⁻¹.

Figure 3

Time course of the cyclopropanation reaction of styrene catalyzed by YfeX. The catalyst was prepared at 20 μM concentration and reduced with 500 equivalents of Na₂S₂O₄ before addition of 1000 equivalents of styrene and 2000 equivalents of EDA at final volume of 500 μL. The organic products were extracted with 3 mL of ethyl acetate after the following time points: 2, 5, 10, 30, and 60 minutes. Finally, the product was quantified by GC/MS. Exponential fit of the data gives a k_obs=0.096 min⁻¹.

Figure 4

(a) Iron‐porphyrin carbene (IPC) complex of YfeX with the substrate dimethylphenylsilane present in the active site. (b) Plot of RMSD for the YfeX IPC bound to dimethylphenylsilane substrate. (c) Plot of the distance between the carbene carbon (C1) and the substrate silicon (Si). (d) Plot of the N4−Fe−C1−C2 dihedral angle (see panel a) during the course of the simulation.

Figure 5

(a) QM/MM potential energy profile for Si−H insertion catalyzed by YfeX‐IPC, calculated at the BS2+ZPE level of theory. (b–c) Geometries of the reactant and product complexes for the OSS state, and (d–f) geometries of the transition states in the OSS, CSS and triplet spin states. The relative energies in (a) are in kcal/mol while the distances in (b–f) are in Å.

Figure 6

Electrostatic potential surface for the active site of (a) YfeX (PDB code 2IIZ) from the outside, and (b) zoomed‐in version for YfeX, (c) Rma TDE (PDB code 6CUN), and (d) Mb (H64V, V68A) (PDB code 6M8F). Electrostatic potential surfaces were drawn in PyMOL using APBS electrostatics (the red color corresponds to negative potential and the blue color corresponds to positive potential).