Abstract
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 fungus Aspergillus flavus, was characterized. Sequence analyses and copper content determination demonstrated Af_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 (KM = 0.16 ± 0.03 mM) than to ABTS (KM = 1.89 ± 0.12 mM). Furthermore, the best fitting 3D-model was based on the ascorbate oxidase from Cucurbita pepo var. melopepo. The laccase-like activity of Af_AO1 on ABTS (Vmax = 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 Af_AO1. According to the biochemical and structural characterization, Af_AO1 was classified as ascorbate oxidase with unusual, laccase-like activity.
Highlights
Ascorbate oxidases catalyze the oxidation of ascorbic acid to dehydroascorbate (DHA), their specific function has not yet been fully elucidated [1,2]
This paper focuses on the characterization of an oxidoreductase from Aspergillus flavus to elucidate its classification within the multi-copper oxidases (MCO) family
We investigated a novel ascorbate oxidase Af_AO1 from A. flavus
Summary
Ascorbate oxidases catalyze the oxidation of ascorbic acid to dehydroascorbate (DHA), their specific function has not yet been fully elucidated [1,2]. The MCO family is one of the earliest and best-studied enzyme groups [8,9] As their name suggests, MCOs utilize copper to oxidize various substrates, concomitantly reducing oxygen to water [10]. The MCO family is highly diverse and includes, besides ascorbate oxidases (EC 1.10.3.3), laccases (EC 1.10.3.2), ceruloplasmin, and ferroxidases (EC 1.16.3.1) [1]. In contrast to laccases, have narrow substrate specificity, almost exclusively oxidizing ascorbic acid to DHA, but can accept other lactone-ring containing substrates [21]. We found that Af_AO1 was highly active on ascorbic acid, and oxidized 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), one of the most commonly used laccase substrates.
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