Computational design of an imine reductase: mechanism-guided stereoselectivity reversion and interface stabilization

Abstract

Imine reductases (IREDs) are important biocatalysts in the asymmetric synthesis of chiral amines. However, a detailed understanding of the stereocontrol mechanism of IRED remains incomplete, making the design of IRED for producing the desired amine enantiomers challenging. In this study, we investigated the stereoselective catalytic mechanism and designed an (R)-stereoselective IRED from Paenibacillus mucilaginosus (PmIR) using pharmaceutically relevant 2-aryl-substituted pyrrolines as substrates. A putative mechanism for controlling stereoselectivity was proposed based on the crucial role of electrostatic interactions in controlling iminium cation orientation and employed to achieve complete inversion of stereoselectivity in PmIR using computational design. The variant PmIR-Re (Q138M/P140M/Y187E/Q190A/D250M/R251N) exhibited opposite (S)-stereoselectivity, with >96% enantiomeric excess (ee) towards tested 2-aryl-substituted pyrrolines. Computational tools were employed to identify stabilizing mutations at the interface between the two subunits. The variant PmIR-6P (P140A/Q190S/R251N/Q217E/A257R/T277M) showed a nearly 5-fold increase in activity and a 12 °C increase in melting temperature. The PmIR-6P successfully produced (R)-2-(2,5-difluorophenyl)-pyrrolidine, a key chiral pharmaceutical intermediate, at a concentration of 400 mM with an ee exceeding 99%. This study provides insight into the stereocontrol elements of IREDs and demonstrates the potential of computational design for tailored stereoselectivity and thermal stability.

Scheme 1. Asymmetric Reduction of Protonated 2-DFPL by IRED.

Fig. 1. (A) Multisequence alignment of reported IREDs with (R)-stereoselectivity towards 2-aryl-substituted pyrrolines. The IREDs used for sequence alignment include PmIR (this study), ScIR (WP_003961038.1), IRED-GF3546 (BAM99301),BcsIRED (WP_000841637),SrIRED (WP_012890722),PeIRED (WP_010497949.1),SaIRED (WP_016644884.1), and NhIRED (WP_017538739.1). The figure was prepared using ESPript (numbering as PmIR). Y187 and D250 were conserved in the (R)-selective enzyme. (B) Representative snapshots showing the reorientation of the D250 side chain. (C) Docking conformation in pro-R pose and following conformation analysis with molecular dynamics (MD) simulations. The distance distributions indicate the high proportion of reactive conformation in the pro-R pose.

Fig. 2. (A) The chirality of the amine produced by PmIR was determined by transferring a hydride ion from NADPH to a prochiral carbon atom on the specific CN face. (B) Superimposition of WT (green) and Y187E (cyan) docking results and a putative mechanism for stereocontrol, in which the electrostatic interaction plays a crucial role in controlling the orientation of the 1-pyrroline face.

Fig. 3. Computational reversion of PmIR stereoselectivity. (A) Flowchart illustrating the process of computational stereoselectivity reversion of PmIR (B) the catalytic geometric constraints based on the pro-S pose TS structure were used in design and conformation sampling. (C) In total, 30 000 variant structures were sorted based on the criteria of a total score <−1645.0 and constraint energy <10.0 kcal mol⁻¹. These structures were further filtered based on an interface energy <−8.0 kcal mol⁻¹, resulting in the selection of the top four non-redundant variant structures. (D) The top variants with different side chain conformations were superimposed to compare their differences. Side-chain conformations of residues generated by virtual mutagenesis are shown in lines. (E) The occurrence of reactive pro-S conformations in the PmIR-Re complex. (F) The occurrence of reactive pro-S conformations in the complex_5352 variant. (G) The occurrence of reactive pro-S conformations in the complex_25658 variant. (H) The occurrence of reactive pro-S conformations in the complex_6645 variant.

Fig. 4. (A) Stabilizing mutations included in PmIR-6P are marked in the sphere. The mutations identified in alanine scanning (P140A/Q190S/R251N) are in red. Residues forming salt bridges are indicated in yellow (R257-D87/E217-R176). The T277M identified using the ΔΔG^fold calculation is green. The radius of gyration for WT and PmIR-6P was compared, indicating a more compact structure for PmIR-6P. (B) The salt bridge of E217-R176 and the distance comparison of backbone Cα between E217 and R176 in WT and PmIR-6P. (C) The salt bridge of D87-R257 and the distance comparison of backbone Cα between D87 and R257 in WT and PmIR-6P.

Fig. 5. Enzymatic activity and stereoselectivity of WT, PmIR-Re, and PmIR-6P toward different 2-aryl-substituted pyrrolines. The relative activity of WT was set as 100% in bar charts except for 2-phenyl-1-pyrroline and 5-(4-fluorophenyl)-3,4-dihydro-2H-pyrrole which set PmIR-Re as 100%.

Fig. 6. Reaction curve of WT and PmIR-6P toward different substrate loadings.