Enzymology of NRPS and PKS proteins by mass spectrometry

通过质谱法对 NRPS 和 PKS 蛋白进行酶学分析

• 批准号: 7028299
• 负责人: PIETER C DORRESTEIN
• 金额: $ 2.14万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-16 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7028299
• 关键词: SDS polyacrylamide gel electrophoresis; acyl carrier protein; alpha benzopyrone; antibiotics; antineoplastics; biological products; biotechnology; cofactor; drug design /synthesis /production; electrospray ionization mass spectrometry; enzyme biosynthesis; enzyme substrate; infrared spectrometry; interferometry; peptide chemical synthesis; polyketide synthase; postdoctoral investigator; posttranslational modifications; protein purification; proteomics

DESCRIPTION (provided by applicant): Electrospray fourier transfer mass spectrometry (ESI-FTMS) provides a powerful tool for analysis of covalent modifications of proteins. This proposal applies ESI-FTMS for the detection of transient covalent modifications of non-ribosomal-petide synthetases (NRPS) and polyketide synthases. Initially, a general protocol will be developed for the rapid identification of the acyl domains. Subsequently, this method will be extended to the identification of the in vivo or in vitro "preferred" substrates for acyl domains. This method will then be applied to the identification of the substrate(s) for the calE8 gene product involved in the biosynthesis of the antitumor agent calicheamycin, a PKS for which we do not know the substrate. Finally, ESI-FTMS will be used for the kinetic characterization of the NRPS found in the biosynthesis of the aminocoumarin antibiotics coumeramycin and clorbiocin while the substrates are still connected to their acyl domains.

描述（由申请人提供）： 电喷雾傅里叶转移质谱 (ESI-FTMS) 为分析蛋白质共价修饰提供了强大的工具。该提案应用 ESI-FTMS 检测非核糖体肽合成酶 (NRPS) 和聚酮化合物合成酶的瞬时共价修饰。最初，将开发一个通用协议来快速识别酰基结构域。随后，该方法将扩展到鉴定酰基结构域的体内或体外“优选”底物。然后，该方法将应用于抗肿瘤剂加利霉素（一种我们不知道其底物的 PKS）生物合成中涉及的 calE8 基因产物的底物的鉴定。最后，ESI-FTMS 将用于氨基香豆素抗生素香豆霉素和氯生菌素生物合成中发现的 NRPS 的动力学表征，同时底物仍与其酰基结构域相连。