Abstract
Cyclodipeptide synthases (CDPSs) can catalyze the formation of two successive peptide bonds by hijacking aminoacyl-tRNAs from the ribosomal machinery resulting in diketopiperazines (DKPs). Here, three CDPS-containing loci (dmt1–3) are discovered by genome mining and comparative genome analysis of Streptomyces strains. Among them, CDPS DmtB1, encoded by the gene of dmt1 locus, can synthesize cyclo(L-Trp-L-Xaa) (with Xaa being Val, Pro, Leu, Ile, or Ala). Systematic mutagenesis experiments demonstrate the importance of the residues constituting substrate-binding pocket P1 for the incorporation of the second aa-tRNA in DmtB1. Characterization of dmt1–3 unravels that CDPS-dependent machinery is involved in CDPS-synthesized DKP formation followed by tailoring steps of prenylation and cyclization to afford terpenylated DKP compounds drimentines. A phytoene-synthase-like family prenyltransferase (DmtC1) and a membrane terpene cyclase (DmtA1) are required for drimentines biosynthesis. These results set the foundation for further increasing the natural diversity of complex DKP derivatives.
Highlights
Cyclodipeptide synthases (CDPSs) can catalyze the formation of two successive peptide bonds by hijacking aminoacyl-tRNAs from the ribosomal machinery resulting in diketopiperazines (DKPs)
NRRL F-5123 and S. aidingensis China General Microbiological Collection Center (CGMCC) 4.5739 following in silico analysis of Streptomyces genomes available on GenBank (Fig. 2)
High-performance liquid chromatography (HPLC) analysis of the culture supernatants showed that five DKP compounds 1–5 were produced by E. coli cells containing dmtB1 (Fig. 3)
Summary
Cyclodipeptide synthases (CDPSs) can catalyze the formation of two successive peptide bonds by hijacking aminoacyl-tRNAs from the ribosomal machinery resulting in diketopiperazines (DKPs). A phytoene-synthase-like family prenyltransferase (DmtC1) and a membrane terpene cyclase (DmtA1) are required for drimentines biosynthesis These results set the foundation for further increasing the natural diversity of complex DKP derivatives. Functional characterization demonstrates that DmtB1 is a CDPS synthesizing cyclo(L-Trp-L-Xaa) (X = Val, Pro, Leu, Ile, or Ala) with cWV being the major product. The underlying molecular basis for substrate specificity of DmtB1 is probed via extensive mutagenesis of the amino acid residues in the two binding pockets. These three CDPSdependent biosynthetic loci are characterized and revealed a CDPS-dependent pathway whereby the DKP product is prenylated and cyclized by tailoring enzymes to afford terpenylated diketopiperazine compounds, dubbed drimentines (DMTs, Fig. 1).
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