Diketopiperazine Formation in Fungi Requires Dedicated Cyclization and Thiolation Domains

Abstract

Cyclization of linear dipeptidyl precursors derived from nonribosomal peptide synthetases (NRPSs) into 2,5diketopiperazines (DKPs) is ac rucial step in the biosynthesis of alarge number of bioactive natural products.However,the mechanism of DKP formation in fungi has remained unclear, despite extensive studies of their biosyntheses.H ere we show that DKP formation en route to the fungal virulence factor gliotoxin requires as eemingly extraneous couplet of condensation (C) and thiolation (T) domains in the NRPS GliP.I n vivo truncation of GliP to remove the CT couplet or just the T domain abrogated production of gliotoxina nd all other gli pathway metabolites.P oint mutation of conserved active sites in the Cand Tdomains diminished cyclization activity of GliP in vitro and abolished gliotoxin biosynthesis in vivo.V erified NRPSs of other fungal DKPs terminate with similar CT domain couplets,s uggesting ac onserved strategy for DKP biosynthesis by fungal NRPSs.

Heterologous and Engineered Biosynthesis of Nematocidal Polyketide–Nonribosomal Peptide Hybrid Macrolactone from Extreme Thermophilic Fungi

“…41 Analysis of the active sites of ThmB-C T and TqaA-C T domains showed that the ThmB-C T domain contains the intact characteristic "HHxxxD" motif of the classical C domain (Figure S6); however, the first His residue is mutated to a Ser residue in the TqaA-C T domain (SHxxxD). 40 Further phylogenetic analysis among fungal hrPKS−C T domains, single-and multimodule NRPS-C T domain, 40,42 diketopiperazine NRPS (e.g., GliP)-C T domain, 43 classical NRPS C domains, as well as ester bond-forming C domains from bacteria 43 showed that the ThmB-C T domain was clustered in a separate clade, implying that it has a new catalytic function (Figure 4A and Figure S6A).…”

Chain release mechanisms in polyketide and non-ribosomal peptide biosynthesis

“…230 The same Cterminal C T -PCP arrangement is found in other NRPSs, including the previously mentioned nanangelenin B synthase NanA, suggesting a conserved function. 182,230 Fig. 27 Structure of a C T domain.…”

“…230 Interestingly, the PCP downstream of the GliA C T domain is also essential for chain release, indicating that it serves to tether the NRP intermediate specically for the C T domain. 230 The same Cterminal C T -PCP arrangement is found in other NRPSs, including the previously mentioned nanangelenin B synthase NanA, suggesting a conserved function. 182,230 Fig.…”

Sulfur-Containing Compounds from Endophytic Fungi: Sources, Structures and Bioactivities

“…Diketopiperazines are expected to be biosynthetically assembled from two amino acid building blocks by nonribosomal peptide synthetases [ 65 ]. On the other hand, the biogenesis of many sulfur-containing compounds remains incompletely understood.…”