Structure-guided electrostatic tuning of subunit interfaces to enhance direct electron transfer and stability in d-fructose dehydrogenase

Oxidoreductases capable of direct electron transfer (DET) are attractive biocatalysts for bioelectrochemical devices because they can transfer electrons directly to electrodes without mediators. Generally, in DET-type reactions, the electron transfer pathway from the catalytic site to the electrode-active site, and subsequently to the electrode, follows a single route. Consequently, rapid intramolecular electron transfer is necessary to enhance the efficiency of DET-type reactions. One possible factor that could decrease the reaction efficiency is structural features of the enzymes such as electrostatic repulsion between subunits. In this study, we rationally engineered d-fructose dehydrogenase (FDH), a multi-subunit DET-type oxidoreductase, to enhance its bioelectrocatalytic performance by modulating electrostatic interactions at the interfaces between catalytic and cytochrome subunits. Based on the structural information, we introduced site-specific mutations by replacing acidic amino acid residues with lysine (a basic residue) to reduce inter-subunit repulsion. The resulting FDH variants exhibited up to a 2-fold increase in DET-type catalytic current density compared with recombinant (native) FDH (rFDH), while maintaining comparable catalytic activity in solution-based assay. In addition, compared with rFDH, the variants were thermally stable and retained higher enzymatic activity after heat treatment. These results indicate that reducing inter-subunit repulsion not only facilitates DET but also enhances the structural robustness of the enzyme. This study demonstrates that the targeted tuning of inter-subunit interactions is an effective strategy for improving both the performance and durability of DET-type oxidoreductases. This approach provides a general design principle for developing next-generation bioelectrocatalysts for biosensors, biofuel cells, and other bioelectronic applications.