A Fungal Ascorbate Oxidase with Unexpected Laccase Activity

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.

Transcriptome Analysis Reveals That C17 Mycosubtilin Antagonizes Verticillium dahliae by Interfering with Multiple Functional Pathways of Fungi

“…Among the upregulated genes, four genes encoding copper ion transmembrane transporters (VDAG_01771, VDAG_06704, VDAG_06151, VDAG_04029) were located on the tonoplast or plasma membrane. Copper is a basic trace element and in a variety of antioxidant enzymes in fungi, including tyrosinase [ 72 ], ascorbate oxidase [ 73 ], uridine nucleosidase, and superoxide dismutase [ 74 , 75 , 76 ]. Copper also participated in antioxidation of cytochrome C and promoted growth and development of the body.…”

Gene Profiling of the Ascorbate Oxidase Family Genes under Osmotic and Cold Stress Reveals the Role of AnAO5 in Cold Adaptation in Ammopiptanthus nanus

“…These works advanced the understanding of the biological functions of plant AOs. However, the previous studies mainly focused on the function of a single AO [ 34 , 35 , 36 ], and a genome-wide analysis of the structure, evolution, and response to abiotic stress of AO is still lacking.…”

Cu Nanoparticles/N-Doped Ti3C2Tx-Based Fluorescence Sensor for Detecting Ascorbic Acid

“…In that, AAOs can catalyze the oxidation reaction of ascorbic acidturning AA to dehydroascorbate (DHA), along with the reduction of O 2 to H 2 O. 2,3 Several researchers propose that AAOs' expressions and selectivity are regulated by auxin and light. 4,5 All of the aforementioned information points out that ascorbate oxidase plays a possible role in signal perception and transduction.…”

“…They are located either at the surface of the cytoplasm or bound to the cell wall. In that, AAOs can catalyze the oxidation reaction of ascorbic acid-turning AA to dehydroascorbate (DHA), along with the reduction of O 2 to H 2 O. , Several researchers propose that AAOs’ expressions and selectivity are regulated by auxin and light. , All of the aforementioned information points out that ascorbate oxidase plays a possible role in signal perception and transduction . It is noteworthy that the function of AAOs can be improved by raising oxygen levels in the absence of light.…”