Unusual 8-formyl-FAD governs activity of Formate Oxidase (FOX) from Aspergillus oryzae

Recently, formate oxidase (FOX; E.C. 1.2.3.1) from Aspergillus oryzae was identified as a member of the glucose-methanol-choline (GMC) oxidoreductase superfamily of enzymes through amino-acid sequence and three-dimensional structural analysis; FOX is the first and only reported member of the GMC enzymes capable of catalyzing the oxidation of an acid substrate. Additionally, WT FOX was shown to exhibit an unusual UV absorption spectrum that was due to a non-covalently bound 8-formyl FAD in place of the typical FAD cofactor present in most GMC oxidoreductases. Although the presence of an enzyme bound 8-formyl FMN has been reported previously as a result of site-directed mutational studies on lactate oxidase (LOX), FOX is the first reported case of 8-formyl FAD being present in a wild-type enzyme. While FAD is bound covalently through an 8α-N^ε2-histdyl linkage has been shown to be important for catalysis in some GMC enzymes, the formation of 8-formyl-FAD in LOX has been shown previously to result in complete inactivation of the enzyme; the presence of 8-formyl-FAD in FOX was proposed to be an artifact. As a result, both the formation and potential role of the 8-formyl-FAD cofactor in formate oxidase was investigated through the use of steady-state kinetics, site-directed mutagenesis, ICP analysis, UV and fluorescence spectrometry, LCMS, and light-exposure studies. Surprisingly, the results from these studies not only indicate that 8-formyl-FAD is present in the active form FOX but that its formation is crucial for activity.