New insights into the disulfide bond formation enzymes in epidithiodiketopiperazine alkaloids.

Epidithiodiketopiperazines (ETPs) are a group of bioactive fungal natural products and structurally feature unique transannular disulfide bridges between α, α or α, β carbons. However, no enzyme has yet been demonstrated to catalyse α, β-disulfide bond formation in these molecules. Through genome mining and gene deletion approaches in Trichoderma hypoxylon, we identified a putative biosynthetic gene cluster of pretrichodermamide A (1), which requires a FAD-dependent oxidoreductase, TdaR, for the irregular α, β-disulfide formation in 1 biosynthesis. In vitro assays of TdaR, together with AclT involved in aspirochlorine and GliT involved in gliotoxin biosynthesis, proved that all three enzymes catalyse not only the conversion of red-pretrichodermamide A (4) to α, β-disulfide-containing 1 but also that of red-gliotoxin (5) to α, α-disulfide-containing gliotoxin (6). These results provide new insights into the thiol-disulfide oxidases responsible for the disulfide bond formation in natural products with significant substrate and catalytic promiscuities.