Identification of Substrate Binding Site of Fungus Derived Flavin Adenine Dinucleotide Dependent Glucose Dehydrogenase (2016)

【Introduction】 The fungi derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (GDHs) are flavoproteins that catalyze the oxidation of first hydroxyl group of glucose and other sugar molecules, using FAD as the primary electron acceptor (systematic name; D-glucose:acceptor 1-oxidoreductase). Recently, fungi-derived FADGDHs have received attention as the enzymes for blood glucose monitoring in diabetes patients, especially for the use in the self-monitoring of blood glucose (SMBG), because of their oxygen insensitivity and relatively narrow substrate specificity, in particular their lack of activity toward maltose. However, unlike glucose oxidase, a gold standard enzyme for blood glucose monitoring, especially for the strictly narrow specificity toward glucose, fungi derived FADGDHs react with xylose as the substrate. We have previously reported on the characterization of Aspergillus flavus derived FADGDH ( Af GDH) from genome database 1 , and also the stabilization of Af GDH by introducing a disulfide bond 2 . Recently we have reported on the first 3D structure of fungus derived FADGDH 3 . In order to construct an engineered enzyme meeting with the current increasing demands in the accuracy of sensors for SMBG, further advance in our understanding of the mechanism of substrate recognition of this enzyme is necessary. In this study, we report the site directed mutagenesis studies toward the residues expected to influence the binding of substrate, based on the structural analyses of Af GDH crystal structure (PDB ID：4ynu). 【Methods】 The residues responsible for the substrate binding and/or recognition of Af GDH and Aspergillus niger derived GOx ( An GOx), were predicted based on the reported crystal structure (PDB ID:4ynu, 1cf3). According to this prediction, several mutant Af GDHs were designed and constructed by the sited directed mutagenesis. Af GDH wild type (WT) and mutants were prepared using recombinant Escherichia coli . The enzyme activities and substrate specificities were determined by measuring dye mediated dehydrogenase activity, spectrometrically, using phenazine methosulphate as the electron mediator and 2,6-dichlorophenolindophenol(DCIP) as the final electron acceptor, and by monitoring decrease in the absorbance of oxidized DCIP at 600nm. 【Results and Discussion】 Several residues existing within the distance which may interact with glucono-δ-lactone by their side chain were observed from the ternary complex structure of Af GDH (4ynu). Assuming that this side chain of each residue may recognize and interact with glucose, Af GDH structure was compared with An GOx. As results, residues, possibly recognize 1 st , 2 nd , 3 rd or 4 th hydroxyl group of glucose are conserved within Af GDH and An GOx. Especially, two residues recognize 1 st hydroxyl group of glucose are completely conserved. Mutational analyses resulted in the complete loss of the catalytic activity, thus suggesting that these residues are the catalytic residues. The residue interacting 4 th hydroxyl group is conserved, and has been recognized that plays an important role in discrimination of monosaccharide from disaccharides, such as maltose. Interestingly, the introduction of mutation on this residue did not result in the significant change in the substrate specificity, especially toward maltose. The results suggested that the predicted residue may have a limited role in the determination of substrate specificity, and other residues may have more crucial roles. Significant difference in the substrate interaction was observed at the position where may recognize 6 th hydroxyl group, which would be the reason of the Af GDH substrate specificity reacting with xylose. Further mutational analyses will be reported. (1) K. Mori, et al ., Biotechnol Lett . 33 (11) 2255-63(2011) (2) G. Sakai, et al ., Biotechnol Lett . 37 (5) 1091-99(2015) (3) H. Yoshida, et al ., Sci. Rep. 5:13498 doi: 10.1038/srep13498(2015)