Reprogramming the Rossmann fold signature motif creates orthogonal redox biocatalysts.
10 affiliations
- 1Department of Chemical and Biomolecular Engineering, University of California, Irvine, Irvine, CA, USA.
- 2Genome Center, University of California, Davis, Davis, CA, USA.
- 3Department of Biological Chemistry, University of California, Irvine, Irvine, CA, USA.
- 4Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA.
- 5Department of Chemistry, University of California, Davis, Davis, CA, USA.
- 6Department of Biochemistry and Molecular Medicine, University of California, Davis, Davis, CA, USA.
- 7Department of Chemical and Biomolecular Engineering, University of California, Irvine, Irvine, CA, USA. [email protected].
- 8Department of Biological Chemistry, University of California, Irvine, Irvine, CA, USA. [email protected].
- 9Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA. [email protected].
- 10UCI Center for Synthetic Biology (CSB), University of California, Irvine, Irvine, CA, USA. [email protected].
Abstract
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.
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BibTeX
@article{ping2026reprogramming,
title = {{Reprogramming the Rossmann fold signature motif creates orthogonal redox biocatalysts.}},
author = {Yu Ping and Jin Young Kim and Minh-Anh L Dinh and Emma Luu and Suphanida Worakaensai and Shiding Zhang and William B Black and Emma Monge and Hans Jefferson C Ng and Sean Perea and Yulai Zhang and Youtian Cui and Edward King and Justin B Siegel and Han Li},
journal = {Nature communications},
year = {2026},
month = sep,
doi = {10.1038/s41467-026-77633-7},
url = {https://europepmc.org/article/MED/42869905},
note = {CC BY},
}
RIS
TY - JOUR
TI - Reprogramming the Rossmann fold signature motif creates orthogonal redox biocatalysts.
AU - Yu Ping
AU - Jin Young Kim
AU - Minh-Anh L Dinh
AU - Emma Luu
AU - Suphanida Worakaensai
AU - Shiding Zhang
AU - William B Black
AU - Emma Monge
AU - Hans Jefferson C Ng
AU - Sean Perea
AU - Yulai Zhang
AU - Youtian Cui
AU - Edward King
AU - Justin B Siegel
AU - Han Li
PY - 2026
DA - 2026/09/12
JO - Nature communications
DO - 10.1038/s41467-026-77633-7
UR - https://europepmc.org/article/MED/42869905
AB - 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.
SN - 2041-1723
ER -