A Fungal Ascorbate Oxidase with Unexpected Laccase Activity

Ascorbate oxidases are an enzyme group that has not been explored to a large extent. So far, mainly ascorbate oxidases from plants and only a few from fungi have been described. Although ascorbate oxidases belong to the well-studied enzyme family of multi-copper oxidases, their function is still unclear. In this study,Af_AO1, an enzyme from the fungusAspergillus flavus, was characterized. Sequence analyses and copper content determination demonstratedAf_AO1 to belong to the multi-copper oxidase family. Biochemical characterization and 3D-modeling revealed a similarity to ascorbate oxidases, but also to laccases.Af_AO1 had a 10-fold higher affinity to ascorbic acid (K-M= 0.16 +/- 0.03 mM) than to ABTS (K-M= 1.89 +/- 0.12 mM). Furthermore, the best fitting 3D-model was based on the ascorbate oxidase fromCucurbita pepovar.melopepo. The laccase-like activity ofAf_AO1 on ABTS (V-max= 11.56 +/- 0.15 mu M/min/mg) was, however, not negligible. On the other hand, other typical laccase substrates, such as syringaldezine and guaiacol, were not oxidized byAf_AO1. According to the biochemical and structural characterization,Af_AO1 was classified as ascorbate oxidase with unusual, laccase-like activity.