DISCOVERY AND CHARACTERIZATION OF PHOSPHONIC ACID BIOSYNTHESIS PATHWAYS

膦酸生物合成途径的发现和表征

基本信息

批准号：
7249591
负责人：
WILLIAM W METCALF
金额：
$ 36.77万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-03-01 至 2012-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7249591
关键词：
Actinobacteria class Actinomyces Address Alanine Amino Acids Anabolism Area Back Binding Biochemical Biogenesis Biological Assay Biological Factors Carbon Categories Class Complement Complex Mixtures Computer software Data Set Defect Detection Development Engineering Enzymatic Biochemistry Enzymes Fourier Transform Gene Cluster Genes Herbicides Human Hybrids Illinois Ions Kinetics Laboratories Libraries Ligase Link Mass Spectrum Analysis Metabolic Methylation Molecular Mutagenesis Mutase Object Attachment Pathway interactions Peptide Synthesis Performance Pharmaceutical Preparations Phosphonic Acids Phosphorus Production Property Refractory Research Research Personnel Role Screening procedure Series System Techniques Time Universities Upper arm analog base desire experience ginsenoside M1 inorganic phosphate instrumentation mass spectrometer metabolomics mutant novel peptide synthase phosphinothricin phosphonate programs scaffold small molecule thioester tool

项目摘要

Compounds with C-P bonds represent an understudied class of natural products with proven and attractive properties as drugs for humans or herbicides. With stable carbon-phosphorus bonds, phosphonates and phosphinates are useful scaffolds that mimic phosphates and display desirable pharmacological properties. This major class of bioactive compounds will be addressed by an intensely collaborative team at the University of Illinois. The Kelleher Laboratory will leverage its core expertise in ultra-high performance Fourier-Transform Mass Spectrometry (FTMS) to implement instrumentation and tailored software for phosphonate compounds and biosynthetic intermediates in both targeted and discovery modes. For known phosphonates, both small molecule MS (metabolomics) and large molecule MS (FTMS-based assays), will be used to interrogate phosphonate intermediates both free and bound as thioesters to non-ribosomal peptide synthetases. Further, the compound K-26 will be linked to its biosynthetic gene cluster using a general fosmid-screening approach employing a highly automated FTMS-based screen. The enzymology underlying the biosynthesis of phosphinothricin tripeptide (PTT) will be elucidated. With emphasis on the PTT system, the Kelleher Laboratory will use its extensive experience in non-ribosomal peptide synthesis to dissect the timing of P- methylation and the role of a curious tandem thiolation domain in the biosynthetic assembly line. Armed with mechanistic understanding of the thiotemplate portion of this gene cluster, a series of phs mutants will be screened using large molecule FTMS to engineer the NRPS portion of the PTT cluster from S. vidriochromogenes, with production of unnatural PTT analogues in a heterologous producer, S. lividans, to follow. For both targeted analysis and discovery, the negative mass defect of phosphorus along with selective MS/MS detection approaches will require tailored software for "phosphonate-directed" metabolomics to filter large datasets emanating from ion trap/Fourier-Transform hybrid mass spectrometers operating at 7, 12, and 14.5Tesla. A particular strength of a MS approach to phosphonate discovery is that new compounds are identified based on structural features and not modes of biosynthesis or spectrum of activity in a bioassay. The FTMS approaches to engineered production and discovery of new phosphonates complements the many other strategies presented in this integratedP01.

具有 C-P 键的化合物代表了一类尚未得到充分研究的天然产物，其经过验证且具有吸引力具有作为人类药物或除草剂的特性。具有稳定的碳磷键、膦酸盐和次膦酸盐是模仿磷酸盐并显示出所需药理学特性的有用支架。这一类主要的生物活性化合物将由该中心的一个密切合作的团队来解决。伊利诺伊大学。凯莱赫实验室将利用其在超高性能傅里叶变换质量方面的核心专业知识光谱测定 (FTMS)，用于实施用于膦酸盐化合物的仪器和定制软件靶向和发现模式的生物合成中间体。对于已知的膦酸盐，两者都小分子 MS（代谢组学）和大分子 MS（基于 FTMS 的检测）将用于询问膦酸酯中间体既可以游离，也可以作为硫酯与非核糖体肽合成酶结合。此外，化合物 K-26 将使用通用的 fosmid 筛选与其生物合成基因簇连接方法采用高度自动化的基于 FTMS 的屏幕。生物合成的酶学基础草胺膦三肽（PTT）的作用将得到阐明。 Kelleher 重点关注 PTT 系统实验室将利用其在非核糖体肽合成方面的丰富经验来剖析P-的时机甲基化和生物合成装配线中奇怪的串联硫醇化结构域的作用。武装有为了理解该基因簇的硫模板部分，一系列 phs 突变体将被使用大分子 FTMS 进行筛选，以设计来自 S. 的 PTT 簇的 NRPS 部分。 vidriochromogenes，在异源生产者 S. lividans 中产生非天然 PTT 类似物，跟随。对于有针对性的分析和发现，磷的负质量缺陷以及选择性 MS/MS 检测方法将需要用于“磷酸盐导向”代谢组学的定制软件来过滤来自离子阱/傅里叶变换混合质谱仪的大型数据集，运行温度为 7、12、和14.5特斯拉。 MS 方法发现膦酸盐的一个特殊优势是新化合物是根据结构特征而不是生物合成模式或活性谱来识别的生物测定。 FTMS 方法用于工程化生产和发现新的膦酸盐补充了本综合 P01 中提出的许多其他策略。