title: Phosphopantetheinylation and specificity of acyl carrier proteins in the mupirocin biosynthetic cluster.
abstract: Acyl carrier proteins are vital for the biosynthesis of fatty acids and polyketides. The mupirocin biosynthetic cluster of Pseudomonas fluorescens encodes eleven type I ACPs embedded in its multifunctional polyketide synthase (PKS) proteins plus five predicted type II ACPs (mAcpA-E) that are known to be essential for mupirocin biosynthesis by deletion and complementation analysis. MupN is a putative Sfp-type phosphopantetheinyl transferase. Overexpression of three type I and three type II mupirocin ACPs in Escherichia coli, with or without mupN, followed by mass spectroscopy revealed that MupN can modify both mupirocin type I and type II ACPs to their holo-form. The endogenous phosphopantetheinyl transferase of E. coli modified mAcpA but not mAcpC or D. Overexpression of the type II ACPs in macp deletion mutants of the mupirocin producer P. fluorescens 10586 showed that they cannot substitute for each other while hybrids between mAcpA and mAcpB indicated that, at least for mAcpB, the C-terminal domain determines functional specificity. Amino acid alignments identified mACPs A and D as having C-terminal extensions. Mutation of these regions generated defective ACPs, the activity of which could be restored by overexpression of the macp genes on separate plasmids.
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Shields
authors.forename: Ayesha S
authors.initials: AS
authors.lastname: Rahman
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Arthur
authors.forename: John
authors.initials: J
authors.lastname: Crosby
authors.forename: Joanne
authors.initials: J
authors.lastname: Hothersall
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Simpson
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Thomas
mesh_terms: D000213:Acyl Carrier Protein / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000096:biosynthesis; D018942:Macrolides / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D016712:Mupirocin / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology; D009154:Mutation; D048630:Polyketide Synthases / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D011551:Pseudomonas fluorescens / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics / Q000378:metabolism*
chemical_list: D000213:Acyl Carrier Protein; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005227:Fatty Acids; D018942:Macrolides; D011994:Recombinant Proteins; C082662:phosphopantetheinyl transferase; D048630:Polyketide Synthases; D016712:Mupirocin; D017855:Transferases (Other Substituted Phosphate Groups)