Identification and characterization of a type II peptidyl carrier protein from the bleomycin producer Streptomyces verticillus ATCC 15003

Du Liangcheng, Ben Shen

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

Background: Nonribosomal peptide synthetases (NRPSs) catalyze the assembly of a structurally diverse group of peptides by the multiple-carrier thiotemplate mechanism. All NRPSs known to date are exclusively type I modular enzymes that consist of domains, such as adenylation (A), peptidyl carrier protein (PCP) and condensation (C) domains, for individual enzyme activities. Although several A and PCP domains have been demonstrated to function independently, aminoacylation in trans has been successful only between PCPs and their cognate A domains. Results: We have identified within the bleomycin-biosynthesis gene cluster from Streptomyces verticillus ATCC15003 the blml gene that encodes a discrete PCP protein. We overexpressed the blml gene in Escherichia coli, purified the Blml protein, and demonstrated that apo-Blml can be efficiently modified into holo-Blml either in vivo or in vitro by PCP-specific 4'-phosphopantetheine transferases (PPTases). Unlike the PCP domains in type I NRPSs, Blml lacks its cognate A domain and can be aminoacylated by Val-A, an A domain from a completely unrelated type I NRPS. Conclusions: Blml represents the first characterized type II PCP. The Blml type II PCP, like the PCP domains of type I NRPSs, can be 4'-phosphopantetheinylated by PCP-specific PPTases but is biochemically distinct in that it can be aminoacylated by an A domain from a completely unrelated type I NRPS. Our results provide for the first time the genetic and biochemical evidence to support the existence of a type II NRPS, which might be useful in the combinatorial manipulation of NRPS proteins to generate novel peptides.

Original languageEnglish (US)
Pages (from-to)507-517
Number of pages11
JournalChemistry and Biology
Volume6
Issue number8
DOIs
StatePublished - Aug 1999

Keywords

  • Biosynthesis
  • Bleomycin
  • Nonribosomal peptide synthetase
  • Peptidyl carrier protein
  • Streptomyces verticillus

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

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