Reprogramming the Rossmann fold signature motif creates orthogonal redox biocatalysts.

Biological reducing power is carried by nicotinamide adenine dinucleotide (phosphate) (NAD(P)/H), which supports cellular functions and cannot be specifically directed to engineered metabolic pathways. Nicotinamide mononucleotide (NMN(H)) has emerged as an orthogonal redox cofactor to address this. Herein, to create NMN(H)-specific enzymes that no longer interact with cellular NAD(P)/H pools, we perturb the ancient, conserved GxGxxG motif in Rossmann fold enzymes that enables persistent NAD(P)/H recognition. We build variants NRC-01 and NRC-02 on phosphite dehydrogenase (PTDH), which eliminate electron leaking to NAD(P)H-dependent side reactions while driving NMNH-dependent biotransformation with ~240-fold higher productivity than existing catalysts. Testing the design principle on 12 other vastly diverse enzymes yields additional NMN(H)-orthogonal enzymes catalyzing valuable biomanufacturing reactions, and reveals a potential rule predicting the translatability of this method. Rosetta modeling, structural alignment, and experimental results reveal that Rossmann fold reprogramming, paired with engineered structural reinforcement, may offer a general route to orthogonal redox biocatalysts.