Engineering Glucose Dehydrogenase to Favor Totally Synthetic Biomimetic Cofactors Containing Carboxyl Group

Abstract The utilization of unnatural nicotinamide cofactors for reactions catalyzed by oxidoreductases has gained increasing interest. Totally synthetic nicotinamide cofactor biomimetics (NCBs) are cost‐effective and convenient to synthesize. Thus, it has become increasingly important to develop enzymes that accept NCBs. Here, we have engineered Ss GDH to favor a newly synthesized unnatural cofactor 3‐carbamoyl‐1‐(4‐carboxybenzyl) pyridin‐1‐ium (BANA + ). Using in situ ligand minimization tool, sites 44 and 114 were identified as hotspots for mutagenesis. All the double mutants demonstrated 2.7–7.7‐fold improvements in catalytic activity, and the best double mutant E44D/E114 L exhibited 10.6‐fold increased catalytic efficiency toward BANA + . These results provide valuable information for the rational engineering of oxidoreductases with versatile NCBs‐dependency, as well as the design of novel biomimetic cofactors.