Escherichia coli CueO Efficiently Detoxifies Aflatoxin B₁ via a Mediator-Enhanced Two-Step Mechanism

Aflatoxin B₁ (AFB₁) 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 AFB₁ into the less toxic aflatoxin Q₁, 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 AFB₁ 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 AFB₁. Mutants (Met510Leu, Asp439Ala, and Pro444Ala) exhibited enhanced enzymatic activity toward ABTS but showed no improvement in the catalytic efficiency for AFB₁ transformation. Molecular docking suggests this is because AFB₁ binds to surface-exposed residue Ser243, distal to the active site. These findings highlight E. coli CueO's a promising candidate for managing AFB₁ contamination.