The presence of dimeric molecule is prevalent among natural products, yet detailed mechanism of their biosynthesis is poorly characterized. Such compounds remain to be a significant challenge to traditional and biomimetic chemical synthesis as well. Thus, we sought to investigate how a C�C bond can be formed between chemically identical molecules enzymatically. We focused on (�)-ditryptophenaline because of its complex chemical structure involving dimerization at unactivated carbon atoms. Since similar structural motif is also found in other indole alkaloid natural products, we felt that our findings here will have a broad impact on our understanding of the mechanism of dimeric natural product biosynthesis.Using the compounds obtained from suitable deletion mutant strains of (�)-ditryptophenaline-producing Aspergillus flavus A1421 as references, we characterized the activity of a P450, DtpC, involved in the ditryptophenaline biosynthesis. This confirmed that DtpC was responsible for cyclization of cyclo-N-methylphenylalanyltryptophanyl and setting up the compound for the ensuing dimerization to form (�)-ditryptophenaline. Furthermore, DtpC exhibited a broad substrate tolerance against diketopiperazines and successfully accepted brevianamide F to generate not only a homodimeric product, (�)-dibrevianamide F, but also a heterodimer of brevianamide F and cyclo-N-methylphenylalanyltryptophanyl, (�)-tryprophenalin. Based on those biochemical studies and the homology-based structural model of DtpC, we propose a biosynthetic mechanism involving concomitant cyclization and dimerization of diketopiperazine via a radical pathway, mediated by the indole ring of the diketopiperazine core.

Cytochrome P450 as dimerization catalyst in diketopiperazine alkaloid biosynthesis

247th National Meeting (2014)

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