Effect of Structure-Guided Single Mutant on Nicotinamide Coenzyme Specificity of Glucose Dehydrogenase from Bacillus Megaterium
: Glucose dehydrogenase (GDH) from Bacillus megaterium IWG3 is a NAD(P)⁺ -dependent oxidoreductase widely used in biosensing and biocatalytic NADPH regeneration. However, its intrinsic preference for NAD⁺ over NADP⁺ limits its application in NADPH -driven processes. Here, we report a structure-guided rational design to invert the coenzyme specificity of GDH by targeting a single residue within the conserved GXXXGXG motif of the Rossmann fold. Molecular docking and structural analysis identified Thr17 as the key residue forming a hydrogen bond with the 2′ - hydroxyl of NAD⁺, thereby discriminating against the 2′ -phosphate of NADP⁺. Three -point mutants—T17G, T17K, and T17R—were constructed, expressed, and kinetically characterized. The T17G mutation dramatically inverted cofactor preference, increasing the catalytic efficiency ratio (NADP⁺/NAD⁺) from 0.78 (wild -type) to 7.5, driven by a 2.4-fold decrease for NADP⁺ and a 4.6 - fold increase in for NAD⁺. Remarkably, the T17K mutant not only shifted preference toward NADP⁺ (specificity ratio 0.96) but also enhanced turnover numbers for both coenzymes by up to 5.2-fold, achieving c atalytic efficiencies of 6.39 mM⁻¹·s⁻¹ (NAD⁺) and 6.15 mM⁻¹·s⁻¹ (NADP⁺) —the highest among all variants tested. In contrast, the T17R mutation severely impaired NADP⁺ binding ( k m = 97.18 mM) and abolished activity. Structural modeling revealed that glycine c reates space to accommodate the 2′ -phosphate, while lysine establishes a favorable electrostatic interaction with the phosphate group; arginine’s bulky guanidinium group causes steric clash. This study demonstrates that a single, rationally designed mutation at position 17 can simultaneously broaden cofactor specificity and improve catalytic efficiency, with the T17K mutant emerging as a superior biocatalyst for NADPH regeneration. The strategy provides a generalizable framework for engineering cofactor preference in short-chain dehydrogenase/reductase family enzymes.