Mechanisms of nonribosomal peptide natural product biosynthesis

描述(由申请人提供)：多肽天然产品代表多种重要的治疗方法。负责构建非核糖体肽的生物合成酶具有复杂的结构，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产生新疗法的一条很有前途的途径。我们的程序是为了阐明非核糖体肽生物合成的关键步骤的机制和提供结构信息。该项目将研究生物合成方法学的两个重要方面：非蛋白来源氨基酸构建块的生物合成以及氨基酸的选择和加载到合成酶机器上。我们将应用X射线结晶学、有机合成和机械酶学的跨学科组合。这一结果将被应用于新型氨基酸构建块的合理设计，也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。选择正在研究的系统既是因为展示了新的酶学，也是因为它在治疗上重要的分子的生物合成中的重要性。该提案描述了三个酶系统，每个系统都为整个项目目标提供了独特的信息。1)非蛋白生成氨基酸是万古霉素类抗生素的关键成分。生物合成3，5-二羟基苯甘氨酸的一个重要步骤是DpgC酶催化的双氧合反应。DpgC是辅因子/金属非依赖性加氧酶中的一员，具有独特的化学和结构。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/抗生素烯二炔类化合物。一种含有稀有辅因子4-亚甲基咪唑酮的新型氨基变位酶SgcC4催化β-酪氨酸的1，2-氨基转移生成β-酪氨酸。3)使用设计的合成类似物作为链接剂，研究非核糖体多肽机械结构域/结构域相互作用的结构基础。我们将把这种方法应用于负责激活和加载氨基酸的独立双域结构。

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.