A sequence motif enables widespread use of noncanonical redox cofactors in natural enzymes.
Noncanonical redox cofactors (NRCs) are low-cost alternatives to the natural redox cofactors nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) for biomanufacturing, offering exquisite electron-delivery control, yet their adoption is limited by the scarcity of compatible enzymes. Screening the aldehyde dehydrogenase (ALDH) family, we identified a conserved RH/QxxR motif that enables widespread NRC activity among natural enzymes. Bos taurus ALDH3a1 exhibits unprecedented turnover with nicotinamide mononucleotide (NMN+), with kcat values exceeding NAD+ and surpassing most engineered NRC-active enzymes by 10-105-fold. Structural analyses reveal that this motif reinforces cofactor positioning and preorganizes the active site independently of the NAD+ adenosine monophosphate moiety. This motif supports activity across simple-synthetic NRCs such as 1-(2-carbamoylmethyl)nicotinamide and, when introduced into diverse ALDH scaffolds, enhances NMN+ activity up to 60-fold. These findings elucidate nature's solution to engineering NRC-active enzymes and offer a blueprint to mine latent evolutionary plasticity in natural enzymes that serve as superior engineering starting points.