Discovery and molecular elucidation of pervasive Aflatoxin B1 oxidation activity in the laccase superfamily

Aflatoxins are mutagenic mycotoxins which are believed to impact over a billion people every year, particularly in the developing world, and, when consumed, lead to stunting, immunosuppression, and liver cancer. With the worsening of climate change, molds and their toxic secondary metabolites will become an even greater risk to humanity. In this work we use in vitro and in silico methodologies to evaluate the potential for and propose the use of multicopper oxidases, specifically laccases, to enzymatically degrade aflatoxin B1 (AFB1), a highly mutagenic mycotoxin, to a safer byproduct. A panel of assays was performed on 45 members of the multicopper oxidase family with 20 different enzymes showing the ability to degrade AFB1 and establishing the pervasiveness of this activity in the family. In order to better understand how laccases oxidize AFB1, we utilized density functional theory to identify likely atoms within AFB1 for oxidation to occur. Once a reaction scheme had been established, we employed computational docking with Rosetta to ascertain which structural features are likely to contribute to AFB1 binding. These predictions can provide insight for future efforts to optimize enzymes for detoxifying AFB1.