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）： 电喷雾傅立叶转移质谱（ESI-FTMS）是一种分析蛋白质共价修饰的有力工具。该提案应用ESI-FTMS检测非核糖体肽合成酶（NRPS）和聚酮酶的瞬时共价修饰。最初，将开发一个通用的协议，用于快速识别的酰基结构域。随后，这种方法将被扩展到在体内或体外的酰基结构域的“优选”底物的鉴定。然后将该方法应用于鉴定参与抗肿瘤剂加利车霉素生物合成的calE 8基因产物的底物，我们不知道底物的PKS。最后，ESI-FTMS将用于在氨基香豆素抗生素香豆素霉素和氯生霉素的生物合成中发现的NRPS的动力学表征，而底物仍然连接到它们的酰基结构域。