A Fungal Ascorbate Oxidase with Unexpected Laccase Activity

Area of Science:

• Biochemistry
• Enzymology
• Mycology

Background:

• Ascorbate oxidases are a poorly understood enzyme group within the multi-copper oxidase family.
• Research has primarily focused on plant and a few fungal ascorbate oxidases, leaving their precise function unclear.
• This study investigates a novel fungal enzyme, Aspergillus flavus ascorbate oxidase 1 (Af_AO1).

Purpose of the Study:

• To biochemically and structurally characterize Af_AO1 from Aspergillus flavus.
• To determine the enzymatic activity and substrate specificity of Af_AO1.
• To classify Af_AO1 within the multi-copper oxidase family and elucidate its function.

Main Methods:

• Sequence analysis and copper content determination to confirm enzyme family.
• Biochemical assays to measure substrate affinity (K) and reaction velocity (V).
• 3D-modeling to predict structural similarities and relationships to known enzymes.

Main Results:

• Af_AO1 belongs to the multi-copper oxidase family, showing high affinity for ascorbic acid (K = 0.16 mM).
• Structural modeling indicates similarity to both ascorbate oxidases and laccases.
• Af_AO1 exhibits significant laccase-like activity on ABTS (V = 11.56 µM/min/mg) but does not oxidize typical laccase substrates like syringaldezine or guaiacol.

Conclusions:

• Af_AO1 is classified as an ascorbate oxidase due to its primary substrate affinity.
• The enzyme possesses unusual, secondary laccase-like activity, broadening the known functional spectrum of fungal ascorbate oxidases.
• This research contributes to a deeper understanding of the diversity and function of multi-copper oxidases in fungi.