Abstract
Amine dehydrogenases (AmDHs) catalyse the conversion of ketones into enantiomerically pure amines at the sole expense of ammonia and hydride source. Guided by structural information from computational models, we create AmDHs that can convert pharmaceutically relevant aromatic ketones with conversions up to quantitative and perfect chemical and optical purities. These AmDHs are created from an unconventional enzyme scaffold that apparently does not operate any asymmetric transformation in its natural reaction. Additionally, the best variant (LE-AmDH-v1) displays a unique substrate-dependent switch of enantioselectivity, affording S- or R-configured amine products with up to >99.9% enantiomeric excess. These findings are explained by in silico studies. LE-AmDH-v1 is highly thermostable (Tm of 69 °C), retains almost entirely its catalytic activity upon incubation up to 50 °C for several days, and operates preferentially at 50 °C and pH 9.0. This study also demonstrates that product inhibition can be a critical factor in AmDH-catalysed reductive amination.
Highlights
Amine dehydrogenases (AmDHs) catalyse the conversion of ketones into enantiomerically pure amines at the sole expense of ammonia and hydride source
imine reductases (IReds) and reductive aminases (RedAms) are normally employed for the synthesis of secondary and tertiary amines[17,18], this property was recently observed in some cases with AmDHs19
The experiment was conducted in the presence of formate dehydrogenase (Cb-FDH) for NADH recycling (Fig. 2a) as described in Methods section (Biocatalytic reductive amination in analytical scale)[23,35]
Summary
Amine dehydrogenases (AmDHs) catalyse the conversion of ketones into enantiomerically pure amines at the sole expense of ammonia and hydride source. Transaminases have proven to be useful and efficient biocatalysts for the stereoselective amination of ketones in laboratory, as well as industrial scale settings[16,20,21]; their downside is the requirement for supra-stoichiometric amounts of an amine donor and/or more enzymes and cofactors[16,20,21,22]. In this context, the AmDH-FDH system enables the efficient reductive amination of prochiral ketones (i.e., TONs > 103) while requiring only HCOONH4 buffer and catalytic NAD23. Other AmDHs have been produced either by domain-shuffling or introducing further mutations into these first generation variants[32,33]
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