An ortho-Quinone Methide Mediates Disulfide Migration in the Biosynthesis of Epidithiodiketopiperazines.

Transannular disulfide functions as a key structural element imparting diverse biological activities of epidithiodiketopiperazines (ETPs). Although previous studies proposed mechanisms, α, β'-disulfide formation in ETPs is not well determined owing to the lack of identification for the hypothetical intermediate. Here, we characterize the key ortho-quinone methide (o-QM) intermediate and prove its involvement in the carbon-sulfur migration from α, α'- to α, β'-disulfide by elucidating pretrichodermamide A biosynthesis, which is catalyzed by a FAD-dependent thioredoxin oxygenase TdaE harboring a noncanonical CXXQ motif. Biochemical investigations of recombinant TdaE and mutants demonstrated that the construction of α, β'-disulfide was initiated by Gln140-triggering proton abstraction for generation of the essential o-QM intermediate, accompanied by β'-acetoxy elimination. Subsequent attack on α, α'-disulfide by Cys137 led to disulfide migration and spiro-furan formation. This study expands the biocatalytic toolbox for transannular disulfide formation and set the stage for targeted discovery of bioactive ETPs.