Identification and characterization of a type II peptidyl carrier protein from the bleomycin producer Streptomyces verticillus ATCC 15003.
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 blmI gene that encodes a discrete PCP protein. We overexpressed the blmI gene in Escherichia coli, purified the BlmI protein, and demonstrated that apo-BlmI can be efficiently modified into holo-BlmI either in vivo or in vitro by PCP-specific 4'-phosphopantetheine transferases (PPTases). Unlike the PCP domains in type I NRPSs, BlmI lacks its cognate A domain and can be aminoacylated by Val-A, an A domain from a completely unrelated type I NRPS.


CONCLUSIONS
BlmI represents the first characterized type II PCP. The BlmI type II PCP, like the PCP domains of type I NRPSs, can be 4'-phospho-pantetheinylated 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.
Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA.
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D000225:Adenine / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001426:Bacterial Proteins; D001483:Base Sequence; D001761:Bleomycin / Q000096:biosynthesis*; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism; D005800:Genes, Fungal / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism
D000596:Amino Acids; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004271:DNA, Fungal; D047908:Intracellular Signaling Peptides and Proteins; C121823:peptidyl carrier protein, type II, Streptomyces verticillus; D001761:Bleomycin; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase; D000225:Adenine