DISCOVERY AND CHARACTERIZATION OF PHOSPHONIC ACID BIOSYNTHESIS PATHWAYS

膦酸生物合成途径的发现和表征

• 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
• 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

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研究的天然产品，具有验证和吸引人 特性作为人类或除草剂的药物。具有稳定的碳磷键，磷酸盐和 磷酸盐是模仿磷酸盐并显示出理想的药理特性的有用脚手架。 这一主要的生物活性化合物将由一个非常合作的团队来解决 伊利诺伊大学。 Kelleher实验室将利用其在超高性能傅立叶转换质量方面的核心专业知识 光谱法（FTMS）以实现用于磷酸化合物的仪器和量身定制的软件 靶向和发现模式中的生物合成中间体。对于已知的磷酸盐，都很小 分子MS（代谢组学）和大分子MS（基于FTMS的测定）将用于询问 膦酸酯中间是硫代酯与非核糖体肽合成酶的硫代植物。 此外，化合物K-26将使用一般的fosmid筛选将其与其生物合成基因簇相关 采用高度自动化的基于FTM的屏幕的方法。生物合成的酶学 将阐明磷脂蛋白三肽（PTT）的磷脂。重点是PTT系统，Kelleher 实验室将利用其在非核糖体肽合成中的丰富经验来剖析P-的时间 甲基化和奇怪的串联硫醇化结构域在生物合成组合线中的作用。武装 对该基因簇的硫型板部分的机械理解，一系列的PHS突变体将是 使用大分子FTM筛选，以从S。 vidriochromogenes，在异源生产者S. lividans中产生不自然的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检测方法将需要针对“磷酸化导向”代谢组学量身定制的软件才能过滤 从离子陷阱/傅立叶转换杂种质谱仪中发出的大型数据集，在7、12， 和14.5tesla。 MS磷酸盐发现方法的特殊优势是新化合物 根据结构特征，而不是生物合成或活性谱的模式鉴定 生物测定。 FTMS的工程生产和发现的方法 补充了该集成P01中提出的许多其他策略。