Cytochrome P450 as Dimerization Catalyst in Diketopiperazine Alkaloid Biosynthesis

Abstract

As dimeric natural products frequently exhibit useful biological activities, identifying and understanding their mechanisms of dimerization is of great interest. One such compound is (−)-ditryptophenaline, isolated from Aspergillus flavus, which inhibits substance P receptor for potential analgesic and anti-inflammatory activity. Through targeted gene knockout in A. flavus and heterologous yeast gene expression, we determined for the first time the gene cluster and pathway for the biosynthesis of a dimeric diketopiperazine alkaloid. We also determined that a single cytochrome P450, DtpC, is responsible not only for pyrroloindole ring formation but also for concurrent dimerization of N-methylphenylalanyltryptophanyl diketopiperazine monomers into a homodimeric product. Furthermore, DtpC exhibits relaxed substrate specificity, allowing the formation of two new dimeric compounds from a non-native monomeric precursor, brevianamide F. A radical-mediated mechanism of dimerization is proposed.

Elucidation of cladofulvin biosynthesis reveals a cytochrome P450 monooxygenase required for anthraquinone dimerization

“…The involvement of cytochrome P450s in the dimerization of napthoquinones, coumarins, and diketopiperazine alkaloid was reported in bacteria, Aspergillus niger, and Aspergillus flavus, respectively (29)(30)(31). In S. coelicolor A3(2), flaviolin is dimerized by a cytochrome P450 (30), and a similar enzyme in Streptomyces griseus dimerizes tetrahydroxynapthalene into 1,4,6,7,9,12-hexahydroxyperylene-3,10-quinone (32).…”

“…In A. niger, the cytochrome P450 KtnC dimerizes dimethyl siderin into kotanin (29). The cytochrome P450 DtpC in A. flavus is responsible for the concomitant pyrroloindole ring formation and homo-or heterodimerization of the diketopiperazine alkaloids ditryptophenaline, dibrevianamide F, and tryptophenaline (31). Nataloe-emodin 10 dimers with alternative aryl-aryl bonds have not been observed in C. fulvum, and they do not have dimeric forms of emodin 5, which suggests that ClaM might be highly selective for both substrate and type of linkage that it catalyzes.…”

Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids

“…[7] and (+)-WIN 64821 [8]. More recently, 1 was found to be produced by another fungus, Aspergillus flavus [9]. To elucidate the biosynthetic pathway of 1, experiments of deficiency of the biosynthetic gene were performed to identify ditryptophenaline biosynthetic gene cluster.…”

“…In the proposed mechanism (Figure 4), a radical formed at N10 through hydrogen atom abstraction by the heme iron of P450 can migrate to C3 with a concomitant C2-N10 ring closure and undergo a radical-mediated coupling with another C3 radical-bearing monomer to form the bridging bond in the dimer like 1. More recently, 1 was found to be produced by another fungus, Aspergillus flavus [9]. To elucidate the biosynthetic pathway of 1, experiments of deficiency of the biosynthetic gene were performed to identify ditryptophenaline biosynthetic gene cluster.…”

“…DtpA, ditryptophenaline nonribosomal peptide synthetase (NRPS); DtpB, N-methyltransferase; DtpC, cytochrome P450; FtmA, fumitremorgin NRPS. More recently, 1 was found to be produced by another fungus, Aspergillus flavus [9]. To elucidate the biosynthetic pathway of 1, experiments of deficiency of the biosynthetic gene were performed to identify ditryptophenaline biosynthetic gene cluster.…”