Reprogramming NAD(P)⁺-Binding Proteins for Iminium Biocatalysis via a Synthetic NAD⁺-Type Cofactor

Area of Science:

• Biocatalysis and protein engineering
• Synthetic cofactor development
• Enzyme mechanism and design

Background:

• Integrating non-natural catalytic functions into proteins is crucial for artificial enzyme design.
• Existing methods face challenges in achieving efficient and selective catalysis with synthetic motifs.

Purpose of the Study:

• To develop a novel NAD+-type synthetic cofactor, aniline adenine dinucleotide (AnAD), for reprogramming protein scaffolds.
• To explore the catalytic capabilities and scope of AnAD in various protein environments.
• To investigate the mechanistic basis of AnAD-mediated catalysis and stereoselectivity.

Main Methods:

• Synthesis of aniline adenine dinucleotide (AnAD) by replacing nicotinamide with an aniline unit.
• Incorporation of AnAD into diverse NAD(P)+-binding protein scaffolds.
• Activity and enantioselectivity assays for tandem Friedel-Crafts alkylation-enantioselective protonation reactions.
• Protein engineering to optimize catalytic performance.
• Mechanistic studies involving active-site loop dynamics.

Main Results:

• AnAD successfully introduced iminium catalysis into various protein scaffolds, enabling new-to-nature reactions.
• Screening revealed broad intrinsic compatibility of natural proteins with AnAD.
• Protein engineering enhanced catalytic activity and stereoselectivity, achieving good substrate generality.
• Mechanistic studies highlighted the role of protein microenvironment and loop dynamics in catalysis and enantioselectivity.

Conclusions:

• AnAD is a versatile synthetic cofactor for reprogramming NAD(P)+-binding proteins.
• This approach enables new-to-nature biotransformations with tunable enantioselectivity.
• The findings demonstrate a general and robust strategy for artificial enzyme design.