A Sequence Motif Enables Widespread Use of Non-Canonical Redox Cofactors in Natural Enzymes

Non-canonical redox cofactors (NRCs) are promising alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)⁺) for biomanufacturing due to low cost and exquisite electron delivery control, yet their adoption is limited by the scarcity of compatible enzymes. Here, we screened the aldehyde dehydrogenase (ALDH) protein family and identified a conserved RH/QxxR sequence motif that enables widespread NRC activity among natural enzymes. Bos taurus ALDH3a1 and Pseudanabaena biceps ALDH exhibit unprecedented turnover with nicotinamide mononucleotide (NMN⁺), with k_cat values matching or exceeding that of NAD⁺ and surpassing most engineered NRC-active enzymes by 10 to 10⁵-fold, based on the relative NRC to native activity. Structural and dynamic analyses reveal this motif reinforces cofactor positioning and pre-organizes the active site without dependence on the adenosine monophosphate moiety of NAD⁺. When introduced into diverse ALDH scaffolds, the RH/QxxR motif enhances NMN⁺ activity up to 60-fold. In addition to NMN⁺, this motif also supports activity across multiple non-nucleotide, simple synthetic NRCs such as 1-(2-carbamoylmethyl)nicotinamide (AmNA⁺). These findings elucidate Nature's solution to the engineering challenge of obtaining NRC-active enzymes and offers a blueprint to mine latent evolutionary plasticity in natural enzymes that serve as superior engineering starting points.