Coenzyme Engineering of Glucose-6-phosphate Dehydrogenase on a Nicotinamide-Based Biomimic and Its Application as a Glucose Biosensor

Nicotinamide adenine dinucleotide (NAD(P)⁺)-dependent oxidoreductases have been widely employed as biocatalysts for numerous applications, such as in vitro biomanufacturing and biosensors. The application of biomimetic nicotinamide coenzymes (BNCs) in an enzymatic redox cascade constitutes a promising alternative that can eliminate the need for expensive natural NAD(P)⁺ coenzymes. Herein, we demonstrated that the coenzyme engineering of glucose-6-phosphate dehydrogenase from Zymomonas mobilis (ZmG6PDH) enhanced its catalytic efficiency (k_cat/K_m) on oxidized nicotinamide mononucleotide (NMN⁺). Compared with the wild-type (WT) enzyme, the optimal mutant R4 exhibited a 112-fold enhancement in catalytic efficiency on NMN⁺, with 4.7 × 10³ and 2.6 × 10³-fold change in the two coenzyme specificity values ([k_cat/K_m]_NMN⁺/[k_cat/K_m]_NAD⁺ and [k_cat/K_m]_NMN⁺/[k_cat/K_m]_NADP⁺), respectively. To obtain insights into the structure–function relationship of the enzyme and its mutant, we determined the crystal structures of WT G6PDH (apo-WT) and the complex of mutant R4 and NMN⁺ (R4:NMN⁺) and performed molecular dynamics simulations of the ternary complexes of the G6PDH/substrate (glucose-6-phosphate, G6P)/coenzyme. The results revealed that the helix region of the coenzyme-binding Rossmann-like domain in mutant R4 was nearer to the active site than the wild-type enzyme, thus creating a more compact substrate/coenzyme-binding site to favor the binding of the substrate G6P and NMN⁺. Furthermore, a glucose biosensor based on the glucokinase (GK)/G6PDH-NMN⁺ biosystem without cryopreservation was constructed using NMN⁺ as the coenzyme. These results indicate that the combination of BNCs and oxidoreductases can be widely employed in biosensors, biocatalysis, and in vitro biomanufacturing.