title: Regeneration of misprimed nonribosomal peptide synthetases by type II thioesterases.
abstract: Nonribosomal peptide synthetases (NRPSs) assemble structurally complex peptides from simple building blocks such as amino and carboxyl acids. Product release by macrocyclization or hydrolysis is catalyzed by a thioesterase domain that is an integrated part of the NRPS enzyme. A second thioesterase of type II (TEII) encoded by a distinct gene associated with the NRPS cluster was previously shown by means of gene disruption to be important for efficient product formation. However, the actual role of TEIIs in nonribosomal peptide synthesis remained obscure. Here we report the biochemical characterization of two such TEII enzymes that are associated with the synthetases of the peptide antibiotics surfactin (TEII(srf)) and bacitracin (TEII(bac)). Both enzymes were shown to efficiently regenerate misacylated thiol groups of 4'-phosphopantetheine (4'PP) cofactors attached to the peptidyl carrier proteins (PCPs) of NRPSs. For TEII(srf), a K(M) of 0.9 microM and a k(cat) of 95 min(-1) was determined for acetyl-PCP hydrolysis. Both enzymes could also hydrolyze aminoacyl or peptidyl PCPs, intermediates of nonribosomal peptide synthesis. However, this reaction is unlikely to be of physiological relevance. Similar intermediates of the primary metabolism such as CoA derivatives and acetyl-acyl carrier proteins of fatty acid synthesis were also not significantly hydrolyzed, as investigated with TEII(srf). These findings support a model in which the physiological role of TEIIs in nonribosomal peptide synthesis is the regeneration of misacylated NRPS, which result from the apo to holo conversion of NRPS enzymes because of the promiscuity of dedicated 4'PP transferases that use not only free CoA, but also acyl-CoAs as 4'PP donors.
authors.affiliation: Biochemie, Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse, D-35032 Marburg, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Schwarzer
authors.forename: Henning D
authors.initials: HD
authors.lastname: Mootz
authors.forename: Uwe
authors.initials: U
authors.lastname: Linne
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Marahiel
mesh_terms: D000213:Acyl Carrier Protein / Q000378:metabolism; D000596:Amino Acids; D001059:Apoproteins / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001414:Bacitracin / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D004789:Enzyme Activation; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D064429:Fatty Acid Synthases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005227:Fatty Acids / Q000096:biosynthesis; D015870:Gene Expression; D006868:Hydrolysis; D007700:Kinetics; D055666:Lipopeptides; D008316:Malonyl Coenzyme A / Q000378:metabolism; D010453:Peptide Synthases / Q000378:metabolism*; D010456:Peptides, Cyclic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012270:Ribosomes; D013869:Thiolester Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014440:Tyrocidine / Q000737:chemistry / Q000378:metabolism
chemical_list: D000213:Acyl Carrier Protein; D000596:Amino Acids; D001059:Apoproteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005227:Fatty Acids; D055666:Lipopeptides; D010456:Peptides, Cyclic; D011993:Recombinant Fusion Proteins; D014440:Tyrocidine; C040157:acpP protein, E coli; D001414:Bacitracin; C009365:surfactin peptide; D008316:Malonyl Coenzyme A; D064429:Fatty Acid Synthases; D013869:Thiolester Hydrolases; C022738:thioesterase II; D054889:Fatty Acid Synthase, Type II; D010453:Peptide Synthases