title: Dissection of the EntF condensation domain boundary and active site residues in nonribosomal peptide synthesis.
abstract: Nonribosomal peptide synthetases (NRPSs) make many natural products of clinical importance, but a deeper understanding of the protein domains that compose NRPS assembly lines is required before these megasynthetases can be effectively engineered to produce novel drugs. The N-terminal amide bond-forming condensation (C) domain of the enterobactin NRPS EntF was excised from the multidomain synthetase using endpoints determined from sequence alignments and secondary structure predictions. The isolated domain was well-folded when compared by circular dichroism to the vibriobactin NRPS VibH, a naturally free-standing C domain. The EntF domain was also fully functional in an assay based on a synthetic small-molecule substrate, seryl N-acetylcysteamine. Active site mutants of the EntF C domain were surprisingly inactive in vitro as compared to their VibH counterparts, yet maintained the overall domain structure. An in vivo assay was developed in the context of the full-length EntF protein to more sensitively probe the activity level of the C domain mutants, and this supported strong effects for the active site mutations. The crucial role of histidine-138 was confirmed by assay of the full-length protein in vitro. These results suggest a strong resemblance of catalysis by the EntF C domain to chloramphenicol acetyltransferase, including an active site organized by an arginine-aspartate salt bridge, a key histidine acting as a general base, and an asparagine instead of a serine stabilizing the proposed tetrahedral intermediate by hydrogen bonding. The precise definition of a functional C domain excised from a NRPS should aid efforts at swapping NRPS domains between assembly lines.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Eric D
authors.initials: ED
authors.lastname: Roche
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Walsh
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003543:Cysteamine / Q000031:analogs & derivatives / Q000737:chemistry; D004758:Enterobactin / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D006639:Histidine / Q000235:genetics; D062385:Hydroxybenzoates / Q000737:chemistry; D008025:Ligases / Q000737:chemistry*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D010452:Peptide Biosynthesis / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D012694:Serine / Q000737:chemistry
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins; D062385:Hydroxybenzoates; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D004758:Enterobactin; D012694:Serine; D006639:Histidine; D003543:Cysteamine; C009135:2,3-dihydroxybenzoic acid; D008025:Ligases; C066234:enterobactin synthetase; D010453:Peptide Synthases; C026269:2,3-dihydroxybenzoate - serine ligase; D000409:Alanine