A Fungal Ascorbate Oxidase with Unexpected Laccase Activity

• Main Authors: Verena Braunschmid, Sarah Fuerst, Veronika Perz, Sabine Zitzenbacher, Javier Hoyo, Cesar Fernandez-Sanchez, Tzanko Tzanov, Georg Steinkellner, Karl Gruber, Gibson S. Nyanhongo, Doris Ribitsch, Georg M. Guebitz
• Format: Article
• Language: English
• Published: MDPI AG 2020-08-01
• Series: International Journal of Molecular Sciences
• Subjects: ascorbate oxidase; laccase; multi-copper oxidase; ABTS
• Online Access: https://www.mdpi.com/1422-0067/21/16/5754
• ISSN: 1661-6596; 1422-0067

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, <i>Af</i>_AO1, an enzyme from the fungus <i>Aspergillus flavus</i>, was characterized. Sequence analyses and copper content determination demonstrated <i>Af</i>_AO1 to belong to the multi-copper oxidase family. Biochemical characterization and 3D-modeling revealed a similarity to ascorbate oxidases, but also to laccases. <i>Af</i>_AO1 had a 10-fold higher affinity to ascorbic acid (<i>K_M</i> = 0.16 ± 0.03 mM) than to ABTS (<i>K_M</i> = 1.89 ± 0.12 mM). Furthermore, the best fitting 3D-model was based on the ascorbate oxidase from <i>Cucurbita pepo</i> var. <i>melopepo</i>. The laccase-like activity of <i>Af</i>_AO1 on ABTS (<i>V_max</i> = 11.56 ± 0.15 µM/min/mg) was, however, not negligible. On the other hand, other typical laccase substrates, such as syringaldezine and guaiacol, were not oxidized by <i>Af</i>_AO1. According to the biochemical and structural characterization, <i>Af</i>_AO1 was classified as ascorbate oxidase with unusual, laccase-like activity.