Schneider A, Stachelhaus T, Marahiel M A
Biochemie/Fachbereich Chemie, Philipps-Universität Marburg, Germany.
Mol Gen Genet. 1998 Feb;257(3):308-18. doi: 10.1007/s004380050652.
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.
对参与肽抗生素非核糖体合成的肽合成酶一级结构的分析揭示了一种高度保守且有序的模块化排列。一个模块至少包含两个结构域，分别参与依赖ATP的底物激活和硫酯形成。这些功能组件的出现和排列与肽产物中所含氨基酸的数量和顺序相关。在本研究中，我们展示了关于枯草芽孢杆菌三模块表面活性素合成酶1（SrfA-A）中亮氨酸激活模块靶向交换的数据。这是通过构建几个杂交srfA-A基因实现的，这些基因通过体内重组被引入表面活性素生物合成操纵子。我们检测了杂交基因的表达，并研究了所得重组肽合成酶的酶活性。我们首次直接证明，来自细菌或真菌的单个最小模块能将其氨基酸特异性活性赋予多模块肽合成酶。此外，研究表明在肽链第二位定向掺入鸟氨酸会引起表面活性素构象的整体改变，并可能导致过早环化或形成分支环状结构。
