Benzimidazoles as competitive inhibitors of FAD-monooxygenase

Benzimidazoles are drugs which target tubulin and are widely used to treat intestinal parasites. Four benzimidazoles are tested with the well-characterized and commercially available bacterial p-hydroxybenzoate hydroxylase (PHBH), which belongs to the group of Class A FAD-monooxygenases, which also includes such enzymes as the FAD-monooxygenase domain of MICAL. PHBH is shown to be competitively inhibited by all four benzimidazoles (mebendazole, albendazole, fenbendazole, and oxibendazole) in the micromolar range in the hydroxylase reaction, but not in the non-physiological NADPH-dehydrogenase reaction of ferricyanide reduction. The inhibition pattern is consistent with benzimidazoles competing with p-hydroxybenzoate for the resting state of the enzyme, indirectly indicating the ordered mechanism of substrate binding. Modeling studies support the conclusions derived from steady-state kinetics.