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Volume 272, Number 8, Issue of February 21, 1997 pp. 4814-4819
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Substrate Specificity of Hybrid Modules from Peptide Synthetases

(Received for publication, October 2, 1996, and in revised form, December 4, 1996)

Andrea Elsner , Heinrich Engert , Wolfram Saenger , Leendert Hamoen ^§ , Gerard Venema ^§ and Frank Bernhard

From the  Freie Universität Berlin, Institut für Kristallographie, Takustrasse 6, D-14195 Berlin, Germany and the ^§ Department of Genetics, University of Groningen, Kerklaan 30, NL-9751 NN Haren, The Netherlands

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.

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