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.
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Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA.
Shen
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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