Flavine
adenine dinucleotide (
FAD) dependent
glucose methanol choline oxidoreductase (GMC
oxidoreductase) is the terminal key
enzyme of the
patulin biosynthetic pathway. GMC
oxidoreductase catalyzes the oxidative ring closure of (E)-
ascladiol to
patulin. Currently, no
protein involved in the
patulin biosynthesis in Penicillium expansum has been experimentally characterized or solved by X-ray diffraction. Consequently, nothing is known about P. expansum GMC
oxidoreductase substrate-binding site and mode of action. In the present investigation, a 3D comparative model for P. expansum GMC
oxidoreductase has been described. Furthermore, a multistep computational approach was used to identify P. expansum GMC
oxidoreductase residues involved in the
FAD binding and in substrate recognition. Notably, the obtained 3D comparative model of P. expansum GMC
oxidoreductase was used for performing a virtual screening of a chemical/
drug library, which allowed to predict new GMC
oxidoreductase high affinity
ligands to be tested in in vitro/in vivo assays. In vitro assays performed in presence of 6-hydroxycoumarin and meticrane, among the highly affinity predicted binders, confirmed a dose-dependent inhibition (17-81%) of
patulin production by 6-hydroxycoumarin (10 µM-1 mM concentration range), whereas the approved
drug meticrane inhibited
patulin production by 43% already
at 10 µM. Furthermore, 6-hydroxycoumarin and meticrane caused a 60 and 41% reduction of
patulin production, respectively, in vivo on apples at 100 µg/
wound.