title: Carrier protein recognition in siderophore-producing nonribosomal peptide synthetases.
abstract: Nonribosomal peptide synthetases (NRPSs) use phosphopantetheine (pPant) bearing carrier proteins to chaperone activated aminoacyl and peptidyl intermediates to the various enzymes that effect peptide synthesis. Using components from siderophore NRPSs that synthesize vibriobactin, enterobactin, yersiniabactin, pyochelin, and anguibactin, we examined the nature of the interaction of such cofactor-carrier proteins with acyl-activating adenylation (A) domains. While VibE, EntE, and PchD were all able to utilize "carrier protein-free" pPant derivatives, the pattern of usage indicated diversity in the binding mechanism, and even the best substrates were down at least 3 log units relative to the native cofactor-carrier protein. When tested with four noncognate carrier proteins, EntE and VibE differed both in the range of substrate utilization efficiency and in the distribution of the efficiencies across this range. Correlating sequence alignments to kinetic efficiency allowed for the construction of eight point mutants of VibE's worst substrate, HMWP2 ArCP, to the corresponding residue in its native VibB. Mutants S49D and H66E combined to increase activity 6.2-fold and had similar enhancing effects on the downstream condensation domain VibH, indicating that the two NRPS enzymes share carrier protein recognition determinants. Similar mutations of HMWP2 ArCP toward EntB had little effect on EntE, suggesting that the position of recognition determinants varies across NRPS systems.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: C Gary
authors.initials: CG
authors.lastname: Marshall
authors.forename: Michael D
authors.initials: MD
authors.lastname: Burkart
authors.forename: Robin K
authors.initials: RK
authors.lastname: Meray
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Walsh
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D017931:DNA Primers; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010204:Pantetheine / Q000031:analogs & derivatives* / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017262:Siderophores / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D017262:Siderophores; D010204:Pantetheine; D010453:Peptide Synthases; C405545:VibF protein, bacteria; C003129:4'-phosphopantetheine