Chemo-enzymatic platform for the discovery of natural and functionalized sactipeptides

抗菌肽是细菌来源的小蛋白家族中的一员。它们的编码序列被组织在基因簇中，这些基因簇编码这些核糖体合成的微型蛋白的合成后修饰所需的酶。它们的特点是至少存在一个分子内硫醚桥，该桥是由半胱氨酸硫醇与伴侣氨基酸的α碳原子连接而形成的。通过这种非常罕见的连接，形成了长度只有几十个氨基酸的生物分子，显示出非凡的化学稳定性和构象刚性。抗菌肽通常具有抗菌活性，因此有可能被用作抗生素。通过数据库挖掘，在细菌基因组中发现了许多可能的活性多肽。只有五个活性多肽的硫醚键的细节是已知的。该项目的主要目标是通过应用分子生物学、生物化学和多肽化学的组合方法对迄今尚未探索的活性多肽进行深入表征。这些知识将被用来利用蛋白质设计和定向蛋白质进化策略来产生具有新功能的活性多肽。到目前为止，还没有描述与疾病相关的靶分子具有量身定做的亲和力的活性多肽。它们成功产生的一个先决条件是详细了解各自的sactionine合成酶对sactionine前体蛋白的分子识别，该酶作为化学-酶法合成非天然sacti多肽的工具。因此，硫醚形成酶识别活性多肽前体的序列和结构参数也将在本项目的框架内进行研究。

Sactipeptides are members of a family of small proteins of bacterial origin. Their coding sequence is organized in gene clusters that encode enzymes required for the postsynthetic modification of these ribosomally synthesized miniproteins. Their hallmark is the presence of at least one intramolecular thioether bridge that is formed by the linkage of a cysteine thiol with the alpha carbon atom of a partner amino acid. Through this very uncommon linkage, biomolecules with only a few dozens of amino acids in length are formed that display extraordinary chemical stability and conformational rigidity. Sactipeptides often show antibacterial activity and therefore are potential candidates for their use as antibiotics. Numerous putative sactipeptides have been identified in bacterial genomes through database mining. Only for five sactipeptides the details of their thioether linkages are known. The main goal of the proposed project is the in depth characterization of up to now unexplored sactipeptides by application of a combination of methods of molecular biology, biochemistry, and peptide chemistry. This knowledge will be used for the generation of sactipeptides with novel functionalities using protein design and directed protein evolution strategies. Sactipeptides with tailor-made affinities towards disease-related target molecules have not been described to date. A prerequisite of their successful generation is a detailed knowledge about the molecular recognition of sactipeptide precursor proteins by the respective sactionine synthase enzyme that serves as tool for the chemo-enzymatic synthesis of non-natural sactipeptides. As a consequence, sequence and structure parameters of sactipeptide precursor recognition by the thioether forming enzymes will also be investigated in the frame of this project.