Reprogramming the Rossmann Fold Signature Motif Creates Orthogonal Redox Biocatalysts.

Area of Science:

• Biocatalysis
• Metabolic Engineering
• Synthetic Biology

Background:

• Biological reducing power is primarily managed by nicotinamide adenine dinucleotide (phosphate) (NAD(P)/H), which poses challenges for directing it to engineered metabolic pathways.
• Nicotinamide mononucleotide (NMN(H)) offers an orthogonal redox cofactor solution, but creating NMN(H)-specific enzymes that avoid cellular NAD(P)/H pools remains difficult.
• Prior enzyme designs preserved the conserved GxGxxG motif in Rossmann fold enzymes, inadvertently maintaining NAD(P)/H recognition.

Purpose of the Study:

• To develop NMN(H)-specific enzymes by engineering the conserved GxGxxG motif in Rossmann fold enzymes.
• To demonstrate the feasibility of creating orthogonal redox biocatalysts independent of cellular NAD(P)/H pools.
• To enhance biocatalytic productivity and cofactor specificity for engineered metabolic pathways.

Main Methods:

• Enzyme engineering by modifying the GxGxxG motif in Rossmann fold enzymes.
• Implementation on phosphite dehydrogenase (PTDH) and glyceraldehyde-3-phosphate dehydrogenase (GapA).
• Utilized Rosetta modeling, structural alignment, and experimental validation in whole cells and cell lysates.

Main Results:

• Engineered PTDH variants (NRC-01, NRC-02) eliminated cofactor "leaking" and increased NMN(H)-dependent biotransformation productivity by ~240-fold.
• Engineered GapA variant (RSQ) achieved a ~2.9x10^4-fold switch in cofactor specificity from NAD+ to NMN+.
• Demonstrated successful creation of active NMN+-specific enzymes by altering the previously essential GxGxxG motif.

Conclusions:

• The conserved GxGxxG motif in Rossmann fold enzymes is mutable, enabling the creation of NMN+-specific enzymes.
• Rossmann fold reprogramming, combined with structural reinforcement, provides a general strategy for developing orthogonal redox biocatalysts.
• This approach overcomes limitations of NAD(P)/H dependency, paving the way for advanced metabolic engineering applications.