Computational Study of Mechanism and Enantioselectivity of Imine Reductase from Amycolatopsis orientalis

Abstract

Imine reductases (IREDs) are NADPH-dependent enzymes (NADPH=nicotinamide adenine dinucleotide phosphate) that catalyze the reduction of imines to amines. They exhibit high enantioselectivity for a broad range of substrates, making them of interest for biocatalytic applications. In this work, we have employed density functional theory (DFT) calculations to elucidate the reaction mechanism and the origins of enantioselectivity of IRED from Amycolatopsis orientalis. Two substrates are considered, namely 1-methyl-3,4-dihydroisoquinoline and 2-propyl-piperideine. A model of the active site is built on the basis of the available crystal structure. For both substrates, different binding modes are first evaluated, followed by calculation of the hydride transfer transition states from each complex. We have also investigated the effect of mutations of certain important active site residues (Tyr179Ala and Asn241Ala) on the enantioselectivity. The calculated energies are consistent with the experimental observations and the analysis of transition states geometries provides insights into the origins of enantioselectivity of this enzyme.

Keywords: biocatalysis; enantioselectivity; imine reductase; reaction mechanism; transition state.

Scheme 1

Reaction catalyzed by imine reductases.

Figure 1

Active site of AoIRED (PDB 5FWN) in complex with NADPH and (R)‐1‐methyl‐1,2,3,4‐tetrahydroisoquinoline (R‐2). Carbon atoms of Tyr179 and Asn241 residues are highlighted in violet.

Scheme 2

Reactions investigated in the present work.

Figure 2

Schematic illustration of the active site model used in the current study, here with 1‐methyl‐3,4‐dihydroisoquinoline (in the protonated form) as the substrate.

Figure 3

Optimized structures of A) the lowest‐energy enzyme‐substrate complexes E:1‐S and E:1‐R , and B) lowest‐energy transition states leading to the two enantiomers, TS‐1‐S and TS‐1‐R . Selected distances are given in Å. Relative energies are indicated in kcal mol⁻¹. For clarity, most of the hydrogens are omitted. Atoms kept fixed during the optimizations are labeled with an asterisk.

Figure 4

Calculated energy profiles for the reaction of substrate 1.

Figure 5

Optimized structures of the lowest‐energy transitions states of A) Tyr179Ala (TS‐1‐S ^Tyr179Ala and TS‐1‐R ^Tyr179Ala ) and B) Asn241Ala (TS‐1‐S ^Asn241Ala and TS‐1‐R ^Asn241Ala ) mutations. Selected distances are given in Å. Relative energies for each pair of TSs are indicated in kcal mol⁻¹. For clarity, most of the hydrogens are omitted. Atoms kept fixed during the optimizations are labeled with an asterisk.

Figure 6

A) Optimized structures of the lowest‐energy enzyme‐substrate complexes E:3‐S and E:3‐R , and B) lowest‐energy transition states leading to the two enantiomers, TS‐3‐S and TS‐3‐R2 . Selected distances are given in Å. Relative energies are indicated in kcal mol⁻¹. For clarity, most of the hydrogens are omitted. Atoms kept fixed during the optimizations are labeled with an asterisk.

Figure 7

Calculated energy profiles for the reaction of substrate 3.