Improved stereocontrol in reductive aminases through steric modification of residues around substrate and cofactor binding pockets†

Asymmetric reductive amination catalysed by reductive aminases (RedAms) provides a green and direct route to 2° and 3° chiral amines. Identifying residues or motifs in these enzymes that facilitate stereocontrol is essential for designing highly desirable enantiodivergent RedAm systems. In this work, we have identified key residues within both the cofactor and substrate binding pockets in a fungal reductive aminase (MaRedAm) and a bacterial imine reductase (AoIRED) that enable stereocontrol through steric modification. In MaRedAm, removing steric bulk at the cofactor binding pocket via W33A or R35A mutation improved (R)-selectivity towards the synthesis of (R)-rasagiline, achieving up to 95% enantiomeric excess (e.e.). Conversely, the W211A mutation at the substrate binding pocket of MaRedAm inverted the stereoselectivity, yielding (S)-rasagiline (42% e.e.). Likewise, varying steric bulk at position N241 in AoIRED allowed for enantiodivergency. Notably, modifying the N241 position significantly improved AoIRED's solution stability and storability. The wild-type enzyme typically precipitates out of solution within 8 h after purification, even when stored at 4 °C, whereas its N241H and N241Y variants remain in solution for up to >1 week. Molecular dynamics (MD) simulations provided detailed insights into the effect of steric modification on stereoselectivity at the cofactor and substrate binding pockets. MaRedAm W33A and W35A mutations induced reorganisation and downsizing of the active site, enhancing (R)-selectivity. In contrast, the W211A mutation enlarged the substrate binding pocket, increasing flexibility for substrate rotation. These findings contribute to the ongoing effort to establish the functional roles of key residues to allow efficient rational engineering of stereoselectivity in IREDs/RedAms.