Pyranose dehydrogenase from Agaricus meleagris (AmPDH), a member of the structural family of GMC oxidoreductases, carries a monocovalently linked FAD cofactor. AmPDH (di)oxidizes many different sugars at C1C4 and is potentially involved in lignin degradation. It is inactive with dioxygen but uses quinones and organometallic compounds instead in its oxidative half-reaction. Functional assignments of active site residues in the enzyme are still elusive to date, making rational protein engineering approaches towards applications in sugar conversions, organic synthesis, and bioelectrochemistry difficult. In the course of this thesis, the monocovalent FAD-linkage and the vicinity of the isoalloxazine in the active site of AmPDH were investigated. Wild type AmPDH and active site variants were heterologously expressed in the yeast Pichia pastoris, purified, and characterized by biochemical, biophysical, and computational means. Experimental techniques included amongst others ECD, TCA/acetone precipitation, GC-MS of reaction products, as well as measuring the oxygen reactivity and redox potential. UV-Vis and stopped-flow spectroscopy were utilized to elucidate steady-state and pre-steady-state kinetics, respectively. Molecular dynamics (MD) simulations and free energy calculations rationalized or suggested experiments.