Mechanisms of nonribosomal peptide natural product biosynthesis

描述（由申请人提供）：肽天然产物代表了不同类别的重要治疗剂。负责构建非核糖体肽的生物合成酶具有复杂的结构架构，并且经常进行困难的化学转化。通过体内工程或化学酶技术操纵生物合成途径是产生新疗法的有前途的方法。提出的是我们的计划，阐明机制，并提供结构信息的关键步骤，在非核糖体肽的生物合成。该项目将研究生物合成方法的两个重要方面：非蛋白质氨基酸构建模块的生物合成以及合成酶机器上氨基酸的选择和装载。我们将应用X射线晶体学，有机合成和机械酶学的跨学科组合。研究结果将应用于新型氨基酸结构单元的合理工程，是我们理解天然产物装配线生物合成复杂机制的长期目标的一部分。选择研究中的系统是因为它们在治疗重要分子的生物合成中既表现出新颖的酶学又具有重要性。该提案描述了三种酶系统，每种酶系统都为整个项目目标提供了独特的信息。1)非蛋白氨基酸是万古霉素类抗生素的关键组分。3，5-二羟基苯甘氨酸生物合成中的一个重要步骤是由酶DpgC催化的双氧化。DpgC是一个非常小的辅因子/金属非依赖性加氧酶组的成员，具有独特的化学和结构。2) ?-氨基酸是广泛的天然和合成化合物（包括抗肿瘤/抗生素烯二炔）中的重要结构单元。一种新的含有稀有辅因子4-亚甲基咪唑酮的氨基变位酶SgcC 4催化？-酪氨酸生成？酪氨酸。3)结构域/结构域的非核糖体肽机制的相互作用的结构基础将使用设计的合成类似物作为拴系剂进行检查。我们将应用这种方法的独立的双结构域结构负责激活和加载的氨基酸。

DESCRIPTION (provided by applicant): Peptide natural products represent a diverse class of important therapeutics. The biosynthetic enzymes responsible for constructing nonribosomal peptides have complex structural architecture and frequently carry out difficult chemical transformations. Manipulation of biosynthetic pathways through in vivo engineering or chemoenzymatic techniques is a promising approach to the generation of novel therapeutics. Presented is our program to elucidate the mechanisms and provide structural information for key steps in the biosynthesis of nonribosomal peptides. The project will examine two important aspects of the biosynthetic methodology: the biosynthesis of nonproteinogenic amino acid building blocks and the selection and loading of amino acids onto the synthetase machinery. We will apply an interdisciplinary combination of X-ray crystallography, organic synthesis and mechanistic enzymology. The results will be applied to the rational engineering of novel amino acid building blocks and is part of our long term goal of understanding the complex mechanisms natural product assembly-line biosynthesis. Systems under study are selected for both exhibited novel enzymology and importance in the biosynthesis of therapeutically important molecules. The proposal describes three enzyme systems, each contributing unique information toward the overall project goals. 1) Nonproteinogenic amino acids are key components of the vancomycin class of antibiotics. An important step in the biosynthesis of 3,5-dihydroxyphenylglycine is the dioxygenation catalyzed by the enzyme DpgC. DpgC is a member of a very small group of cofactor/metal independent oxygenases and has unique chemistry and structure. 2) ?-Amino acids are important building blocks in a wide range of natural and synthetic compounds, including the antitumor/antibiotic enediynes. A novel aminomutase, SgcC4, containing the rare cofactor 4-methylideneimidazolone catalyzes the 1,2-amino shift of ?-tyrosine to generate ?-tyrosine. 3) The structural basis of domain/domain interactions of nonribosomal peptide machinery will be examined using designed synthetic analogs as tethering agents. We will apply this approach to the stand-alone didomain constructs responsible for activation and loading of amino acids.