Actinomycetes-derived imine reductases with a preference towards bulky amine substrates

Abstract

Since imine reductases (IREDs) were reported to catalyze the reductive amination reactions, they became particularly attractive for producing chiral amines. Though diverse ketones and aldehydes have been proved to be excellent substrates of IREDs, bulky amines have been rarely transformed. Here we report the usage of an Increasing-Molecule-Volume-Screening to identify a group of IREDs (IR-G02, 21, and 35) competent for accepting bulky amine substrates. IR-G02 shows an excellent substrate scope, which is applied to synthesize over 135 amine molecules as well as a range of APIs' substructures. The crystal structure of IR-G02 reveals the determinants for altering the substrate preference. Finally, we demonstrate a gram-scale synthesis of an analogue of the API sensipar via a kinetic resolution approach, which displays ee >99%, total turnover numbers of up to 2087, and space time yield up to 18.10 g L^-1 d^-1.

Fig. 1. Synthesis of chiral amines by IREDs.

a IREDs are capable of catalyzing imine formation and imine reduction to generate a wide variety of chiral amines. b The reported relative activities of AspRedAms towards propargylamine and larger amines piperidine, benzylamine, and aniline.

Fig. 2. Screening for IREDs using the increasing-molecule-volume-screening approach.

The number of enzyme hits is shown above the structure of the corresponding secondary amine. The black color scale in each tile is representative of the number of hits generated through the screening with the given substrate. The standard assay mixture (50 μL) contained 5.0 mM ketone, 5.0 mM amine, 1.0 mM NADP⁺, GDH (0.2 mg ml⁻¹), D-glucose (30 mM), 100 mM potassium phosphate buffer (pH 7.0), and 1 mg mL⁻¹ IREDs. The conversion of reductive amination products was determined by comparison with UV-Vis absorptions of synthesized standard reference materials. The enzymes (IR-G02, 21, and 35) with red triangle block are potent IREDs.

Fig. 3. Applying IREDs for analytical-scale synthesis of APIs′ substructures.

a Reductive aminations of 4 with B, D, and E. b Reductive aminations of 3 with B and C. c Reductive amination of 5 with F. d Reductive aminations of 1, 5, 6, and 7 with C and G. e Reductive aminations of 2 and 6 with C, D, and H. f Reductive aminations of 8 and 9 with I.

Fig. 4. Structural and mutagenesis data of IR-G02.

a Apo- structure of IR-G02 (PDB: 7XE8, Supplementary Data 1) and the residues inside the active pocket at the interface. b Analysis of the conversions of the imine reductions and reductive aminations with wild-type and mutants of IR-G02. c Kinetic data of IR-G02 wild-type and mutants W187A, H247A, and M217A for ketone 1. N.D. was not determined because of the low activity.

Fig. 5. Structural analysis for the active-site pocket of IR-G02.

a IR-G02 showed a strong negative electrostatic surface potential in the active-site pocket. b Active site of IR-G02 in complex with NADP⁺ (PDB: 7XR5, Supplementary Data 2). The distance between the residues (T102, W187 and H247) and C4 of NADP⁺ is given in Ångstroms. c, d Analysis of the side opening of WT and mutant D241A of IR-G02. The diameters of the bottleneck are given in ångströms.