Reprogramming the Rossmann fold signature motif creates orthogonal redox biocatalysts.

Computational and experimental study on engineering Rossmann fold enzymes for orthogonal redox cofactor specificity

“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.”

Engineered variants of phosphite dehydrogenase (PTDH) and 12 other diverse Rossmann fold oxidoreductases

“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.”

13 total enzyme scaffolds (PTDH plus 12 others) with kinetic data from three independent replicates

“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.”

Mutating the second glycine in the GxGxxG motif with a partner mutation selectively disrupts NAD(P)/H binding while retaining NMN(H) activity

“Our work here shows that mutating the second G to a flexible and hydrogen bonding-capable residue (G → Q or G → R) offers a way to selectively disrupt NAD(P)/H activity while still allowing NMN(H) activity.”

Engineered PTDH variants achieved significantly higher productivity for NMNH-dependent biotransformation compared to existing catalysts

“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.”

The design principle successfully translated to 12 diverse enzymes with strict GxGxxG motifs, but failed in scaffolds with divergent motifs

“In enzymes bearing a strict GxGxxG Rossmann signature, paired mutation of the second glycine and an interaction partner consistently shifts activity away from NAD(H)/NADP(H) and toward NMN(H)-dependent catalysis; in contrast, scaffolds with divergent Rossmann motifs are less permissive, highlighting the precision of engineering needed at this conserved motif.”

Engineered enzymes exhibit weaker absolute activity using NMN(H) compared to wild-type enzymes using natural cofactors

“In this work, the NMN(H)-orthogonal enzymes still have much weaker activity using NMN(H), compared to the wild-type enzymes using natural cofactors (Table 1).”