NADPH-dependent Secondary Amine Organocatalysis hosted by a Nucleotide-binding Domain

The concept of organocatalysis has been applied to facilitate “new-to-nature” reaction modes via artificial enzyme design. However, it remains challenging to recruit structurally complex natural molecules as synthetic reagents. Here, we have reported a generic design strategy that allows generation of a NADPH-dependent hybrid catalyst whose action is orchestrated by a secondary amine; this system recruits a reaction mode not commonly seen among enzymes, whilst involving an intricate cofactor that cannot be used by existing organocatalysts. A secondary amine organocatalytic motif was incorporated into protein scaffolds as an unnatural amino acid by expansion of the genetic code. When introduced into the multidrug binding protein LmrR, a hybrid catalyst accepting α,β-unsaturated carbonyl substrates for transfer hydrogenation was established but was confined to the much-simplified biomimetic benzyl dihydronicotinamide (BNAH). Conversely, dihydrofolate reductase (DHFR) contains a nucleotide binding domain and can be converted into a hybrid catalyst that favourably uses NADPH for reaction, thus highlighting the importance of choosing an appropriate scaffold. The DHFR-hosted system tolerates a range of aldehyde substrates and can be coupled with an enzymatic NADPH regeneration scheme. The presented engineering approach can be readily extended to other protein scaffolds for use of different natural molecules in non-natural reaction modes.