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

BackgroundNonribosomal 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.ResultsWe 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.ConclusionsBlml 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