Yohei Suzuki, Yuki Kitazumi, Osamu Shirai, Keisei Sowa
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.