PMID:12564937
| Citation |
Roche, ED and Walsh, CT (2003) Dissection of the EntF condensation domain boundary and active site residues in nonribosomal peptide synthesis. Biochemistry 42:1334-44 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1021/bi026867m |
| Keywords |
Alanine/genetics; Amino Acid Sequence; Amino Acids/chemistry; Amino Acids/genetics; Binding Sites/genetics; Catalysis; Cysteamine/analogs & derivatives; Cysteamine/chemistry; Enterobactin/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Genetic Complementation Test; Histidine/genetics; Hydroxybenzoates/chemistry; Ligases/chemistry; Molecular Sequence Data; Multienzyme Complexes/chemistry; Mutagenesis, Site-Directed; Peptide Biosynthesis/genetics; Peptide Fragments/chemistry; Peptide Fragments/genetics; Peptide Fragments/metabolism; Peptide Synthases/chemistry; Peptide Synthases/genetics; Peptide Synthases/metabolism; Protein Structure, Tertiary/genetics; Serine/chemistry |
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