Enzymology of NRPS and PKS proteins by mass spectrometry

描述(由申请人提供)： 电喷雾傅里叶变换质谱仪(ESI-FTMS)为分析蛋白质的共价修饰提供了强有力的工具。这项建议应用ESI-FTMS来检测非核糖体-肽合成酶(NRP)和聚酮合成酶的瞬时共价修饰。最初，将开发一种通用的方案来快速识别酰基结构域。随后，该方法将被扩展到在体内或体外鉴定酰基结构域的“首选”底物。然后，该方法将被应用于抗肿瘤药物Calicheamcin的生物合成中涉及的calE8基因产物的底物(S)的鉴定，该PKS我们不知道底物。最后，ESI-FTMS将被用于氨基香豆素类抗生素香豆素和氯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。

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.