Abstract
We report the first three-dimensional structure of fungus-derived glucose dehydrogenase using flavin adenine dinucleotide (FAD) as the cofactor. This is currently the most advanced and popular enzyme used in glucose sensor strips manufactured for glycemic control by diabetic patients. We prepared recombinant nonglycosylated FAD-dependent glucose dehydrogenase (FADGDH) derived from Aspergillus flavus (AfGDH) and obtained the X-ray structures of the binary complex of enzyme and reduced FAD at a resolution of 1.78 Å and the ternary complex with reduced FAD and D-glucono-1,5-lactone (LGC) at a resolution of 1.57 Å. The overall structure is similar to that of fungal glucose oxidases (GOxs) reported till date. The ternary complex with reduced FAD and LGC revealed the residues recognizing the substrate. His505 and His548 were subjected for site-directed mutagenesis studies, and these two residues were revealed to form the catalytic pair, as those conserved in GOxs. The absence of residues that recognize the sixth hydroxyl group of the glucose of AfGDH, and the presence of significant cavity around the active site may account for this enzyme activity toward xylose. The structural information will contribute to the further engineering of FADGDH for use in more reliable and economical biosensing technology for diabetes management.
Highlights
Because of the broad substrate specificity of PQQGDH, which can lead to potentially fatal errors in glucose sensing[3], fungus-derived FADGDH is becoming the most popular and reliable enzyme for glucose sensing
We report the first 3D structure of fungus-derived glucose dehydrogenase using flavin adenine dinucleotide (FAD) as a cofactor in complex with LGC
There are two more distinct groups of FADGDHs according to their origin
Summary
Because of the broad substrate specificity of PQQGDH, which can lead to potentially fatal errors in glucose sensing[3], fungus-derived FADGDH is becoming the most popular and reliable enzyme for glucose sensing. Despite the long history of fungus-derived FADGDHs, no 3D structural information is currently available. Such a structure is required for further structure-based, state-of-the-art biomolecular engineering studies of this industrially significant enzyme. We obtained the X-ray structures of binary complex of enzyme and reduced FAD at a resolution of 1.78 Å, and the ternary complex with reduced FAD and D-glucono-1,5-lactone (LGC) at a resolution of 1.57 Å. These structures revealed the overall structure similarity with GOx and the active site structure with residues responsible for FAD and substrate binding. We discuss the detailed structural differences and similarities between FADGDH and GOx, including residues responsible for the difference between a dehydrogenase and an oxidase
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