The bacterial flavin adenine dinucleotide (FAD) glucose dehydrogenase (GDH) complex is composed of a catalytic subunit which contains an FAD cofactor and Fe/S cluster in its redox center, showing catalytic activity, a hitchhiker protein subunit functioning as the bacterial TAT secretion system, and a membrane-bound subunit with three heme c moieties that is responsible for the transfer of electrons between the active-site cofactor and external electron acceptors.<br/><br/>The FADGDH complex has the potential to directly transfer electrons (DET) to an electrode because of the presence of the heme c subunit, and is recognized as DET-type FADGDH. The catalytic subunit belongs to the family of glucose-methanol-choline (GMC) oxidoreductase family. The issue with this enzyme is that the enzyme does not have enough substrate specificity towards glucose as compared to other glucose sensing enzymes such as glucose oxidase. The recent elucidation of its X-ray structure revealed the unique substrate binding cavity of this enzyme. Unlike other GMC-oxidoreductase family glucose oxidoreductases, DET-type FADGDH catalytic subunit is characterized with a large cavity in the active site, and fewer number of residues, which are responsible for the recognition of glucose. The mutations have been designed through the crystal structure of DET-type GDH ( PDB: 6A2U) and then engineered into the wild type enzyme. The strategy of mutation and the characterization of mutant enzymes, which show drastic decrease of enzyme activity toward sugars other than glucose, will be presented.

Engineering of substrate specificity of direct electron transfer type glucose dehydrogenase complex which has a unique substrate binding cavity

ACS Fall 2021

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