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药物。负责构建非核糖体肽的生物合成酶具有复杂的结构结构，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一种有前途的新治疗方法。提出了我们的计划，以阐明机制和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氨基酸的选择和装载到合成酶机器上。我们将应用跨学科的结合x射线晶体学，有机合成和机械酶学。该结果将应用于新型氨基酸构建模块的合理工程，并且是我们理解天然产品装配线生物合成复杂机制的长期目标的一部分。所研究的系统被选择为既表现出新的酶学和在治疗重要分子的生物合成中的重要性。该提案描述了三种酶系统，每一种都为整个项目目标提供了独特的信息。1)非蛋白质原性氨基酸是万古霉素类抗生素的关键成分。生物合成3,5-二羟基苯基甘氨酸的一个重要步骤是由DpgC酶催化的双氧合反应。DpgC是一个非常小的辅因子/金属不依赖加氧酶的成员，具有独特的化学和结构。2)吗?氨基酸是广泛的天然和合成化合物的重要组成部分，包括抗肿瘤/抗生素烯二炔。一种新的氨基转换酶SgcC4，含有罕见的辅助因子4-甲基咪唑酮，催化？-酪氨酸生成？-酪氨酸3)非核糖体肽机制的结构域/结构域相互作用的结构基础将使用设计的合成类似物作为栓系剂进行研究。我们将把这种方法应用于负责激活和装载氨基酸的独立双结构域结构体。

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.