title: In vitro reconstitution of the Pseudomonas aeruginosa nonribosomal peptide synthesis of pyochelin: characterization of backbone tailoring thiazoline reductase and N-methyltransferase activities.
abstract: During iron starvation the Gram-negative pathogenic bacterium Pseudomonas aeruginosa makes the nonribosomal peptide siderophore pyochelin by a four protein, 11 domain assembly line, involving a cascade of acyl-S-enzyme intermediates on the PchE and PchF subunits that are elongated, heterocyclized, reduced, and N-methylated before release. Purified PchG is shown to be an NADPH-dependent reductase for the hydroxyphenylbisthiazoline-S-PchF acyl enzyme, regiospecifically converting one of the dihydroheterocyclic thiazoline rings to a thiazolidine. The K(m) for the PchG protein is 1 microM, and the k(cat) for throughput to pyochelin is 2 min(-1). The nitrogen of the newly generated thiazolidine ring can be N-methylated upon addition of SAM, to yield the mature pyochelin chain still tethered as a pyochelinyl-S-PchF at the PCP domain. A presumed methyltransferase (MT) domain embedded in the PchF subunit catalyzes this N-methylation. Mutation of a conserved G to R in the MT core motif abolishes MT activity and subsequent chain release from PchF. The thioesterase (TE) domain of PchF catalyzes hydrolytic release of the fully mature pyochelinyl chain to produce the pyochelin siderophore at a rate of 2 min(-1), at least 30-40-fold faster than in the absence of hydroxyphenylbisthiazolinyl-COOH (HPTT-COOH) chain reduction and N-methylation. A mutation in the PchF TE domain does not catalyze autodeacylation and release of the pyochelinyl-S-enzyme. Thus, full reconstitution of the nonribosomal peptide synthetase assembly line by purified protein components has been obtained for production of this tandem bisheterocyclic siderophore.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: H M
authors.initials: HM
authors.lastname: Patel
authors.forename: C T
authors.initials: CT
authors.lastname: Walsh
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics / Q000378:metabolism*; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D010636:Phenols / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D017262:Siderophores / Q000096:biosynthesis*; D013844:Thiazoles / Q000378:metabolism; D013869:Thiolester Hydrolases / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C103416:PchD protein, Pseudomonas aeruginosa; D010636:Phenols; D017262:Siderophores; D013844:Thiazoles; C025316:pyochelin; D000587:Oxidoreductases Acting on CH-NH Group Donors; C423621:PchG protein, Pseudomonas aeruginosa; D008780:Methyltransferases; D013869:Thiolester Hydrolases; D010453:Peptide Synthases; C401840:aryl-4,2-bisthiazoline synthetase, Pseudomonas aeruginosa; C104385:non-ribosomal peptide synthase