title: Biosynthesis of novel Pyoverdines by domain substitution in a nonribosomal peptide synthetase of Pseudomonas aeruginosa.
abstract: Pyoverdine is a fluorescent nonribosomal peptide siderophore made by fluorescent pseudomonads. The Pseudomonas aeruginosa nonribosomal peptide synthetase (NRPS) PvdD contains two modules that each incorporate an l-threonine residue at the C-terminal end of pyoverdine. In an attempt to generate modified pyoverdine peptides, we substituted alternative-substrate-specifying adenylation (A) and peptide bond-catalyzing condensation (C) domains into the second module of PvdD. When just the A domain was substituted, the resulting strains produced only wild-type pyoverdine-at high levels if the introduced A domain specified threonine or at trace levels otherwise. The high levels of pyoverdine synthesis observed whenever the introduced A domain specified threonine indicated that these nonnative A domains were able to communicate effectively with the PvdD C domain. Moreover, the unexpected observation that non-threonine-specifying A domains nevertheless incorporated threonine into pyoverdine suggests that the native PvdD C domain exhibited stronger selectivity than these A domains for the incorporated amino acid substrate (i.e., misactivation of a threonine residue by the introduced A domains was more frequent than misincorporation of a nonthreonine residue by the PvdD C domain). In contrast, substitution of both the C and A domains of PvdD generated high yields of rationally modified pyoverdines in two instances, these pyoverdines having either a lysine or a serine residue in place of the terminal threonine. However, C-A domain substitution more commonly yielded a truncated peptide product, likely due to stalling of synthesis on a nonfunctional recombinant NRPS template.
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Calcott
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.
authors.forename: Jeremy G
authors.initials: JG
authors.lastname: Owen
authors.affiliation: Biochemistry Department, University of Otago, Dunedin, New Zealand.
authors.forename: Iain L
authors.initials: IL
authors.lastname: Lamont
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand Centre for Biodiscovery, Victoria University of Wellington, Wellington, New Zealand Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland, New Zealand david.ackerley@vuw.ac.nz.
authors.forename: David F
authors.identifier: http://orcid.org/0000-0002-6188-9902
authors.initials: DF
authors.lastname: Ackerley
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D009842:Oligopeptides / Q000096:biosynthesis* / Q000737:chemistry; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; D013912:Threonine; C042453:pyoverdin; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; C091136:pyoverdine synthetase protein, Pseudomonas