title: Targeted alteration of the substrate specificity of peptide synthetases by rational module swapping.
abstract: Analysis of the primary structure of peptide synthetases involved in the non-ribosomal synthesis of peptide antibiotics has revealed a highly conserved and ordered modular arrangement. A module contains at least two domains, involved in ATP-dependent substrate activation and thioester formation. The occurrence and arrangement of these functional building blocks is associated with the number and order of the amino acids incorporated in the peptide product. In this study, we present data on the targeted exchange of the leucine-activating module within the three-module surfactin synthetase 1 (SrfA-A) of Bacillus subtilis. This was achieved by engineering several hybrid srfA-A genes, which were introduced into the surfactin biosynthesis operon by in vivo recombination. We examined the hybrid genes for expression and investigated the enzymatic activities of the resulting recombinant peptide synthetases. For the first time, we demonstrate directly that an individual minimal module, of bacterial or fungal origin, confers its amino acid-specific activity on a multi-modular peptide synthetase. Furthermore, it is shown that directed incorporation of ornithine at the second position of the peptide chain induces a global alteration in the conformation of surfactin and may result in premature cyclization or a branched cyclic structure.
authors.affiliation: Biochemie/Fachbereich Chemie, Philipps-Universität Marburg, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Schneider
authors.forename: T
authors.initials: T
authors.lastname: Stachelhaus
authors.forename: M A
authors.initials: MA
authors.lastname: Marahiel
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods; D055666:Lipopeptides; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism*; D010456:Peptides, Cyclic; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D055666:Lipopeptides; D010456:Peptides, Cyclic; C009365:surfactin peptide; D010453:Peptide Synthases