No abstract

Homologous modules from two different peptide synthetases were analyzed for functionally equivalent regions. Hybrids between the coding regions of the phenylalanine-activating module of tyrocidine synthetase and the valine-activating module of surfactin synthetase were constructed by combining the two reading frames at various highly conserved consensus sequences. The resulting DNA fragments were expressed in Escherichia coli as C-terminal fusions to the gene encoding for the maltose-binding protein. The fusion proteins were purified, and the amino acid specificities, the acceptance of different nucleotide analogues, and the substrate binding affinities were analyzed. We found evidence for a large N-terminal domain and a short C-terminal domain of about 19 kDa within the two modules, which are separated by the sequence motif GELCIGG. The two domains could be reciprocally transferred between the two modules, and the constructed hybrid proteins showed amino acid adenylating activity. Hybrid proteins fused at various consensus motifs within the two domains were inactive, indicating that the domains may fold independently and represent complex functional units. The N-terminal domain was found to be responsible for the amino acid specificity of the modules, and it is also involved in the recognition of the ribosyl and the phosphate moieties of the nucleotide substrate. For tyrocidine synthetase I, we could confine the sites for amino acid specificity to a region of 330 residues. The C-terminal domain is essential for the enzymatic activity and has a strong impact on the specific activity of the modules.Tyrocidine synthetase and surfactin synthetase are multifunctional peptide synthetases produced by Bacillus brevis and Bacillus subtilis, respectively (1-5). The enzymes belong to a superfamily of adenylate-forming enzymes (6 -8). Small peptides like tyrocidine A and surfactin are formed by a nonribosomal pathway according to the thio-template mechanism (6, 9). Prior to incorporation, amino acids and related compounds are activated as adenylates by cleavage of ATP and release of pyrophosphate. Peptide synthetases exhibit a modular structure with several linked modules of about 100 kDa (1, 10 -16). Each module is responsible for recognition, activation, and incorporation of a specific amino acid constituent into the peptide product. Various modules of peptide synthetases have been sequenced, and several highly conserved motifs were found (7,8,11,14,(17)(18)(19). Mutagenesis and cross-linking experiments gave evidence for the involvement of most of these motifs in the binding of ATP and the adenylate forming activity (20 -23). A serine residue in the highly conserved sequence motif LGG(H/D)S at the C terminus of the modules was clearly identified as the site for covalent attachment of a phosphopantetheine cofactor (24 -26). The deletion of the cofactor attachment site in the phenylalanine-activating modules of tyrocidine synthetase I and gramicidin S synthetase I did not affect the amino acid adenylate forming activity (...