Non-ribosomal peptide synthesis

这个项目致力于了解非核糖体肽合成和非核糖体肽合成酶(NRP)中涉及的剪裁酶的生化和结构方面，包括那些超出经典共线性规则的酶。我们想要研究在库茨奈德组装过程中与C？碳官能化和哌嗪酸合成相关的底物修饰酶。此外，我们将用我们成熟的无线电-高效液相-质谱仪检测方法(全球基因组挖掘方法)筛选含有编码有趣的NRPS或修饰酶序列的细菌菌株，以鉴定由热细胞梭菌、橙色虹吸菌和红多孢菌产生的新的NRPS。我们将从代表来自高温假单胞菌和橙色假单胞菌的一个新的NRPS产品类别的原型的环状糖基化糖二肽的生物合成装配线的生化特征开始，并平行地分析来自5.erythraea的一个不寻常的色氨酸卤化反应的生物合成途径。在这项建议的第三部分，我们想要对几个NRPS模块进行密集的结构研究，采用一种新的化学PCP捕获方法，利用不可水解性氨基辅酶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。

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.