Enzymatic Dimerization of Fungal Natural Products through Intermolecular Disulfide Bridges

Abstract The biosynthetic pathway for the production of natural products from various species often include a dimerization step which increases the structural variability and range of biological activities. These dimerization reactions are highly interesting for biocatalytic applications. Epipolythiodioxopiperazines (ETPs) are an important class of natural products whose potent bioactivities rely on disulfide bridges. ETPs often carry a transannular disulfide bridge and can be linked through C−C and C−N bonds to form dimers. In this study, we shed light on the mechanism of disulfide formation in the case of the dimeric vertihemiptellides (vhe) produced by the fungus Verticillium hemipterigenum BCC1449. These compounds feature a unique intermolecular disulfide bridge which links two monomers together. Using comparative genomics, we identified the 12‐membered vhe gene cluster. In vitro assays, kinetic analysis, and protein mutagenesis, enabled us to characterize VheT, the oxidase catalyzing an intermolecular disulfide bond formation resulting in ETP dimers. We showed that the CxxC motif of VheT plays a crucial role in catalyzing this reaction. VheT is an oxidase linking natural product monomers through intermolecular S−S bridges and could be developed into a biocatalyst for thiol specific dimerization reactions.