The bacterial flavin adenine dinucleotide (FAD) glucose dehydrogenase (FADGDH) is composed of three subunits; 1) a catalytic subunit which belongs to the glucose-methanol-choline (GMC) oxidoreductase family contains an FAD cofactor and Fe/S cluster in its redox center, 2) a hitchhiker protein subunit functioning in the bacterial TAT secretion system, and 3) a membrane-bound subunit with three heme c moieties, responsible for the transfer of electrons between the active-site cofactor and external electron acceptors. The presence of the electron transfer subunit makes this enzyme able to transfer electron directly to the electrode, thereby designates this enzyme as a direct electron transfer (DET) type GDH.<br/>The recent X-ray crystal structure elucidation of its catalytic subunit complexed with small subunit (PDB: 6A2U), revealed the unique substrate binding cavity of this enzyme. Unlike other GMC-oxidoreductase family glucose oxidoreductases, the 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 issue with this enzyme in the application for glucose monitoring is that the enzyme does not have strict substrate specificity towards glucose, especially that it reacts with galactose, an epimer at the 4<sup>th</sup> hydroxy group of glucose, although FAD dependent glucose oxidase or fungi derived FADGDHs do not. Upon the industrial requirement and based on the available structural information, mutations were strategically designed and investigated for their impact on both substrate specificity and catalytic efficiency. Our constructed and recombinantly prepared mutant enzymes showed drastic improvement in substrate specificity, by discriminating galactose from glucose, indicating the molecular recognition of the catalytic subunit of this enzyme was altered. The molecular dynamic simulations of these mutant enzymes’ catalytic site with substrates are currently in underway. These computational simulations will be used to bridge our strategy for designing future mutations that will allow high activity towards glucose while reducing the activity of galactose and other sugar substrates; thereby, we will hone the substrate specificity of DET-type FADGDH for optimum use in future biochemical investigations.

Structural basis understanding of substrate specificity within catalytic subunit of direct electron transfer type FAD glucose dehydrogenase

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