FAD binding and dissociation in GMC-oxidoreductases.

The glucose-methanol-choline (GMC)-oxidoreductase superfamily comprises a large group of flavoenzymes such as glucose oxidase, glucose dehydrogenase and cellobiose dehydrogenase, which have been extensively studied and applied in biocatalysis and biosensors. Since the applicability of recombinant flavoenzymes is compromised by divergent glycosylation patterns and substoichiometric FAD occupancy, this study employed experimental and computational methods to analyze the deflavination and reconstitution of three GMC-oxidoreductases from a structural perspective. The results demonstrated that the amount of glycosylation of flavoenzymes is critical for both processes. FAD dissociation constants for glucose oxidase, glucose dehydrogenase and cellobiose dehydrogenase were determined by three different methods, showing Kd values in the range of 10 to 47 nM. Both, the presence of FAD and N-glycosides increase the thermal stability of the flavoenzymes. Steered molecular dynamics simulations revealed differences in the FAD binding of the three enzymes and indicated an undiscovered route of the FAD to dissociate from GMC-oxidoreductases by movement of a loop-and-lid structure on the enzyme surface. This work provides new insights into the mechanism of FAD binding and dissociation in GMC-oxidoreductases and offers strategies for their recombinant production.