(J-363) Engineering of the direct electron transfer (DET) type glucose dehydrogenase to improve DET ability in the absence of an electron transfer subunit

Continuous glucose monitoring (CGM) systems allow users to receive real-time feedback of their blood-glucose levels to effectively treat and manage their diabetes, therefore play a central role in glycemic control for diabetic patients¹. These CGM systems utilize enzymes that can catalyze the oxidation of glucose, and the ideal electrochemical principle of these enzymes is direct electron transfer (DET). The most prominent DET-type enzyme is the bacterial flavin adenine dinucleotide (FAD) glucose dehydrogenase (GDH) enzyme, which is comprised of three subunits: a catalytic subunit (α) harboring an FAD and 3Fe-4S cluster; a hitchhiker protein (γ) necessary to functionally produce the α-subunit; and an electron transfer subunit (β) harboring three heme c domains capable of direct electron transfer (DET) with an electrode and no external mediators are needed. To achieve a much lower oxidation potential for monitoring, the improvement of the DET-ability from the Fe-S cluster in the catalytic subunit is expected. Currently, this Fe-S cluster shows lower efficiency compared with the enzyme complex containing the β–subunit. In this study, DET-ability of the γα complex was investigated to analyze their potential application on CGM sensors. On the basis of the observation, we introduced mutations based on the current available enzyme structures^2,3.

The wild type γα complex, the wild type γαβ complex, or formerly published γαβ complex harboring truncated β-subunit⁴ was immobilized on the gold electrode by using self-assembled monolayer (SAM) and their DET ability was investigated. By utilizing the elucidated x-ray crystal structure of the wild type (WT) γα complex GDH (PDB: 6A2U) and γαβ complex (PDB:8HDD), mutations were rationally designed proximate to the 3Fe-4S cluster, and mutated enzymes’ electron transfer ability towards external electron acceptors was evaluated.

Electrochemical evaluations revealed that the γα complex has a lower onset potential of catalytic current compared with the γαβ complex by cyclic voltammetry measurements. This result indicates a lower operational potential required for the γα complex in a sensor application. However, the onset potential was higher than the electrode with γαβ complex harboring the truncated β-subunit, which is inconsistent with the anticipated redox potential of the Fe-S cluster and hemes. Hypothesizing that the high onset potential in the DET of the γα complex is due to the Fe-S cluster’s inability to access the electrode, we introduced mutations in the γα complex based on recent structural observations of its quaternary structure suggesting the γα complex² changes its conformation upon binding with the β-subunit³. These mutated enzymes’ electron transfer ability towards external electron acceptors was evaluated by both dye-mediated dehydrogenase activity and electrochemically. Electrochemical characteristics of the γα complex will be compared with the γαβ complex and former published γαβ complex harboring truncated β-subunit⁴.

References

1. Inyoung et al., Biosens.Bioelectron., Vol.181, 2021, 113054,

2. Yoshida et al., Acta Crsyt. (2019) D 75, 841–851

3. Okuda-Shimazaki et al., Comm.Biol., 2022, 5:1334

4. Okuda-Shimazaki et al., Electrochim.Acta, 2018, 277, 276-286,