New insights into the disulfide bond formation enzymes in epidithiodiketopiperazine alkaloids

Abstract

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 <i>Trichoderma hypoxylon</i>, we identified a putative biosynthetic gene cluster of pretrichodermamide A (<b>1</b>), which requires a FAD-dependent oxidoreductase, TdaR, for the irregular α, β-disulfide formation in <b>1</b> biosynthesis. <i>In vitro</i> 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 (<b>4</b>) to α, β-disulfide-containing <b>1</b> but also that of red-gliotoxin (<b>5</b>) to α, α-disulfide-containing gliotoxin (<b>6</b>). 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.