Glucose Dehydrogenase Engineered for the Noncanonical Coenzyme NMN Outperforms Its Natural NAD-Dependent Activity
Year: 2026
Type: preprint
Abstract: Engineering redox enzymes to utilize noncanonical coenzymes is critical for advancing in vitro enzymatic biocatalysis and in vivo bioorthogonal chemistry. However, engineered NAD(P)-dependent dehydrogenases had universally underperformed on small noncanonical coenzymes compared to their wild-type (WT) counterparts on natural NAD(P). Here we developed an Iterative (one-site) Mutagenesis and Evolutionary Strategy (IMES) to evolve a flavin-free, NAD-dependent glucose dehydrogenase (GDH) on nicotinamide mononucleotide (NMN + ). By integrating rational design, directed evolution, and a novel high-throughput screening method, we obtained the variant M3 (E44H/P191T/V187M). For the first time, an engineered dehydrogenase achieved catalytic efficiencies with NMN + that surpassed those of the WT enzyme with its natural coenzyme NAD + . Specifically, variant M3 exhibited a 3.2-fold higher specific activity and a 2.06-fold greater catalytic efficiency ( k cat / K m ) with NMN + than the WT GDH with NAD + . Furthermore, we established functional NMN-recycling cascades by coupling the NMNH-generating M3 variant with four NMNH-consuming redox enzymes, including flavin-free alcohol dehydrogenase and mannitol dehydrogenase, as well as two flavin-dependent enzymes (i.e., an oxidase and a transhydrogenase). This study establishes an NMN-based cascade redox system applicable to both in vitro biocatalysis and in vivo bioorthogonal chemistry. Replacing NAD with the smaller, lower-cost NMN coenzyme greatly reduced the economic burden of cofactor supply, paving the way for more efficient and cost-effective enzymatic biocatalysis.
Source: ChemRxiv
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7214763390