Dissection of the EntF Condensation Domain Boundary and Active Site Residues in Nonribosomal Peptide Synthesis

• Published in: Biochemistry (Easton) 2003-02, Vol.42 (5), p.1334-1344
• Main Authors: Roche, Eric D, Walsh, Christopher T
• Format: Article
• Language: English
• Subjects: 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
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi026867m

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.