Non-ribosomal peptide synthesis

该项目致力于了解非核糖体肽合成和非核糖体肽合成酶（NRPS）中剪裁酶的生化和结构方面，包括那些超越经典共线性规则的酶。我们想研究在库兹纳里德组装过程中与c ß-碳功能化和哌酸合成相关的底物修饰酶。此外，我们将使用我们已经建立的无线电- hplc - ms检测方法（全球基因组挖掘方法）筛选含有编码感兴趣的NRPSs或修饰酶的序列的菌株，以鉴定由热细胞梭菌，金疱疹菌和红糖多孢子菌产生的新型NRPSs。我们将从代表C. thermocellum和H. aurantiacus新NRPS产品类别原型的环糖基化糖二肽的生物合成流水线的生化表征开始，并平行分析5中的生物合成途径。一种不寻常的色氨酸卤化的赤藓。在本提案的第三部分，我们希望通过采用一种新的化学PCPtrapping method，利用不可水解的氨基辅酶a衍生物以及催化亚基之间的共价键来限制结晶过程中的结构域运动，对几个NRPS模块进行深入的结构研究。

This project is dedicated towards the understanding of the biochemical and structural aspects of tailoring enzymes involved in non-ribosomal peptide synthesis and non-ribosomal peptide synthetases (NRPS), including those who go beyond the classical co-linearity rules. We would like to investigate substrate modifying enzymes associated with Cß-carbon functionalization and piperazic acid synthesis during kutznerides assembly. Furthermore, we will screen bacterial strains containing sequences encoding for interesting NRPSs or modifying enzymes with our well established radio-HPLC-MS detection method (global genome mining approach) to identify novel NRPs produced by Clostridium thermocellum, Herpetosiphon aurantiacus and Saccharopolyspora erythraea. We will start with the biochemical characterization of the biosynthetic assembly line for a cyclic glycosylated sugardipeptide that represents an archetype of a new NRPS product class from C. thermocellum and H. aurantiacus and analyze in parallel a biosynthesis pathway from 5. erythraea for an unusual tryptophan halogenation. In the third part of this proposal we would like to perform intense structural studies on several NRPS modules by employing a new chemical PCPtrapping method utilizing non-hydrolysable amino-Coenzyme A derivatives as well as covalent linkages between catalytic subunits to restrict the domain motion during crystallization.