In this study, the stabilization of heterotrimeric FAD dependent glucose dehydrogenase (FADGDH) is reported, which was achieved by designing and introducing inter-subunit disulfide bonds. The direct electron transfer (DET) type FADGDH is a heterotrimeric enzyme, which is composed of three subunits; a hitchhiker protein (γ), a catalytic subunit harboring FAD/3Fe4S cluster (α), and a three heme cs containing electron transfer subunit (β). This heterotrimeric structure is very unique among GMC oxidoreductase family. The presence of electron transfer subunit makes this enzyme capable to transfer electron directly to the electrode which is recognize as a DET-type enzyme. Our former achievement revealed that the γα complex structure (PDB:6a2u) harbors the intrinsic disulfide bond between α and γ subunit, which contributes the stabilization of γα complex. Besides, DET type FADGDH showed two phase thermal inactivation, where the first phase is due to the dissociation of β subunit from γαβ complex. This was confirmed by the chemical crosslinking of γαβ complex with glutaraldehyde brought the high thermal stability. Therefore, the stability of this heterotrimeric enzyme is strongly dependent on the interaction of β subunit with γα complex. These observations encouraged us to introduce the inter-subunit disulfide bonds to improve the stability of this enzyme. In this study, based on recently elucidated partial structure of DET type GDH heterotrimer complex, amino acid substitutions were designed to form the inter-subunit disulfide bonds. From the constructed several mutants, we identified the positions where Cys residues should be introduced. As the results, mutant with newly introduced cysteine residues were recombinantly prepared as the soluble heterotrimer complex, without losing its catalytic activity. The formation of disulfide bonds were confirmed by SDS-PAGE analyses by comparing the migrated bands before and after the reduction of protein samples. Thus designed and prepared Cys-substituted FADGDH showed drastic increase in the thermal stability, which was not inactivated under 65 °C of incubation for more than 30 min. Electrochemical investigation suggested that the Cys-substituted FADGDH kept its characteristic DET ability to the electrode, indicating that thus designed engineered DET type FADGDH gives promise the future application for the continuous glucose monitoring system.

Stabilization of a heterotrimeric FAD dependent glucose dehydrogenase by introducing inter-subunit disulfide bonds

ACS Fall 2021

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.