Applied biocatalysis

Generation of novel amine dehydrogenases with unique catalytic behavior from a ε-deamination lysine dehydrogenase

 Vasilis Tseliou¹, Tanja Knaus¹, Marcelo F. Masman¹, Maria L. Corrado¹, Francesco G. Mutti^1
1 Van ‘t Hoff Institute for Molecular Sciences, HIMS-Biocat, University of Amsterdam, Science Park 904, 1098 XH, The Netherlands. E-mail: V.Tseliou@uva.nl

The increasing number of approved active pharmaceuticals ingredients (APIs) that contain optically active amine moieties, has stimulated the development of new and efficient enzymatic methodologies for the asymmetric synthesis of this class of compounds[1]. In this context, native[2] or engineered amine dehydrogenases (AmDHs)[3] enable the atom-efficient synthesis of α-chiral amines starting from carbonyl-containing compounds. Native AmDHs are solely active on aliphatic ketones, whereas engineered AmDHs—obtained using an identical engineering strategy on similar starting scaffolds—resulted in variants possessing similar and limited activities towards aromatic substrates⁴. Herein, using a computationally-guided approach, we engineered new AmDHs starting from an enzyme that does not exhibit any apparent stereoselectivity for its natural reaction. The best variant (LE-AmDH-v1) is highly thermostable (Tm: 69 °C), retains its catalytic activity upon incubation for 7 days (i.e., residual >99% at RT and 80% at 50 °C) and shows optimal catalytic performance at 50 °C and pH 9.0. LE-AmDH-v1 displays a remarkably extended substrate scope compared to existing AmDHs, thus affording pharmaceutically relevant amines starting from acetophenone and derivatives thereof, α-chromanone, α-tetralone, indanone, etc. Elevated conversions, high productivities and enantiomeric excesses up to >99.9% were obtained. The applicability of LE-AmDH-v1 in organic synthesis was demonstrated in preparative scale aminations (>99.7% conv., >99.7% ee). Although other AmDHs were reported to aminate acetophenone, LE-AmDH-v1 was a superior catalyst with this substrate because of its reduced competitive product inhibition (e.g., 20-fold higher IC₅₀), which led to 15-fold higher catalytic performance (k_app/K_M^eff). A unique substrate-dependent stereo-switchable selectivity is another feature of LE-AmDH-v1 as 6-oxo heptanoic acid was quantitatively aminated with S-selectivity (>99% ee), whereas substrates devoid of the terminal carboxylic moiety gave the R-configured enantiomer (>99% ee). Finally, in silico studies provided insight into the role of the mutations upon ligand binding and explained the enantioselective preferences of LE-AmDH-v1.

[1] Patil, M. D., Grogan, G., Bommarius A., Yun, H. ACS Catal. 2018, 8, 10985-11015.