Escherichia coli CueO Efficiently Detoxifies Aflatoxin B 1 via a Mediator-Enhanced Two-Step Mechanism

Aflatoxin B 1 (AFB 1 ) is a highly toxic mycotoxin that threatens global food and feed safety. While enzymatic detoxification is a promising strategy, robust and efficient enzymes remain scarce. This study identified that the multicopper oxidase CueO from Escherichia coli ( E. coli ) CG1061 transforms AFB 1 into the less toxic aflatoxin Q 1, exhibiting optimal activity at pH 8 and 60 °C. The CueO-2,2′-azino-bis (3-ethylbenzothazoline-6-sulfonic acid) (ABTS) mediator system achieved >90% of AFB 1 transformation in 10 min and complete transformation in 20 min, significantly outperforming CueO alone (51% in 60 min). Mechanistically, CueO oxidizes ABTS to form the ABTṠ + radical, which subsequently oxidizes AFB 1 . Mutants (Met510Leu, Asp439Ala, and Pro444Ala) exhibited enhanced enzymatic activity toward ABTS but showed no improvement in the catalytic efficiency for AFB 1 transformation. Molecular docking suggests this is because AFB 1 binds to surface-exposed residue Ser243, distal to the active site. These findings highlight E. coli CueO’s a promising candidate for managing AFB 1 contamination.