Structural Based Investigation of Artificial Electron Acceptor Interaction of Fungus Derived Flavin Adenine Dinucleotide Dependent Glucose Dehydrogenase
Authors: Okurita Madoka , Yoshida Hiromi , Mori Kazushige , Katsuhiro Kojima , Sode Koji 
Published: February 2020

[Introduction]
Self-monitoring of blood glucose (SMBG) is inevitable for the glycemic control of the diabetic patients, and plays an indispensable role in the current diabetes management. The current most popular principle for the sensor for SMBG is based on the so-called “second generation” type that are the use of the dye mediated dehydrogenase reactions. The principle employs the artificial electron acceptor and monitoring the concentration/quantity of thus reacted (reduced) artificial electron acceptor on the electrode. Fungi-derived flavin adenine dinucleotide dependent glucose dehydrogenases (FADGDHs) have recently been paid significant attention, focusing its narrow specificity, especially as the enzyme not active toward maltose. However, interaction between FADGDHs and artificial electron acceptor has not yet been investigated. Recently, we have reported on the first 3D structures of FADGDH, using Aspergillus flavus derived FADGDH (AfGDH). Here we report the investigation of interaction between mediator and enzyme by elucidating the 3D structure of AfGDH complex with potassium ferricyanide. Mutagenesis studies were also carried out to investigate the role of residues elucidated to interact with ferricyanide.
[Methods]
The structural gene of AfGDH was inserted into an expression vector and the enzyme was recombinantly expressed using E.

coli BL21 (DE3). Soluble AfGDH was harvested and purified by hydrophobic interaction chromatography and anion exchange chromatography. Purified protein was concentrated, and crystallized by sitting-drop vapor-diffusion method. In order to obtain a ternary complex structure with FAD and ferricyanide, a single crystal was soaked in the potassium ferricyanide solution. X-ray diffraction data were collected and structure was determined. Based on the 3D structure, several mutants were constructed by site-directed mutagenesis and evaluated GDH activity, which was determined using either phenazine methosulphate/2,6-dichlorophenol indophenol (PMS/DCIP) or potassium ferricyanide as the electron acceptors.
[Results & Discussion]
A ternary complex structure of AfGDH with FAD and ferricyanide complex was obtained. Two potassium ferricyanide molecules were observed in the protein structure. One potassium ferricyanide interacted with one lysine residue, whereas the other molecules interacted with two lysine residues. In order to elucidate the role of these lysine residues, mutant AfGDHs were constructed by substituting lysine residues with alanine. The structural gene of AfGDH mutants were inserted into an expression vector and the enzyme was recombinantly prepared using E. coli BL21 (DE3). All mutants were expressed at soluble fraction. These mutants were subjected for anion exchange chromatography for purification. Interestingly, the mutant enzymes were eluted at different salt concentration than those eluted in the purification of wild type enzyme, suggesting that surface net charge was significantly changed by these mutations. Some of the mutant AfGDH showed unique property in the specificity toward electron acceptors. The GDH activities were compared by using either PMS/DCIP or ferricyanide as the electron acceptor, for both wild type and mutant enzymes. Although the alanine substitutions to these lysine residues did not significantly influenced PMS/DCIP based dye mediated GDH activity, but some mutation affected the ratio of dye mediated GDH activities of those using PMS/DCIP and of using ferricyanide. These results, together with the results of the change in the elution properties during anion exchange chromatography, suggested the lysine residues, we focused, play important role in the affinity toward ferricyanide during oxidative half reaction.
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