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首页> 外文学位 >Active site mapping of iterative polyketide synthases and the detection of polyketide intermediates using high-resolution Fourier transform mass spectrometry.
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Active site mapping of iterative polyketide synthases and the detection of polyketide intermediates using high-resolution Fourier transform mass spectrometry.

机译：迭代聚酮化合物合成酶的活性位点图谱和使用高分辨率傅里叶变换质谱法检测聚酮化合物中间体的位置。

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摘要

In an attempt to better understand the biosynthesis of the enediyne core of the antitumor compound C-1027 by SgcE and to investigated the iterative steps involved in the formation of the methylbutyrl side-chain of lovastatin by LovF, high resolution mass spectrometry was used to identify peptides containing domain active sites of these polyketide synthases and to determine with very high mass accuracy the mass of substrates covalently attached to the active site. In particular the phosphopantetheinyl ejection assay was used to successfully detect the formation of the methylbutyrate by LovF on the ACP active site. It was found that methylbutyrate is detectable only at very low substrate concentrations, but complete formation of the product was carried out by LovF. SgcE was found to be active and capable of catalyzing elongation of a polyene intermediate. The substrate loading and one iteration of elongation were detected using mass spectrometry and the phosphopantetheinyl ejection assay. The results from these experiments showed the power of high-resolution mass spectrometry in studying the mechanisms of action by which these iterative polyketide synthases function and form their fully elongated products.
机译：为了更好地了解SgcE合成抗肿瘤化合物C-1027的烯二炔核的生物合成，并研究通过LovF形成洛伐他汀的甲基丁缩醛侧链的迭代步骤，采用了高分辨率质谱法含有这些聚酮化合物合酶的结构域活性位点的肽，并以非常高的质量精度确定共价附着于活性位点的底物的质量。特别地，使用磷酸泛肽基团喷射测定法成功地检测了ACP活性位点上LovF形成的丁酸甲酯。发现丁酸甲酯仅在非常低的底物浓度下才可检测到，但是通过LovF完成了产物的完全形成。发现SgcE是活性的并且能够催化多烯中间体的伸长。使用质谱法和磷酸泛肽基喷射测定法检测底物负载和伸长的一次迭代。这些实验的结果显示了高分辨率质谱法在研究这些迭代聚酮化合物合酶起作用并形成其完全伸长的产物的作用机理中的作用。

著录项

  • 作者

    Meehan, Michael Jospeh.;

  • 作者单位

    University of California, San Diego.;

  • 授予单位 University of California, San Diego.;
  • 学科 Chemistry Analytical.;Chemistry Biochemistry.
  • 学位 M.S.
  • 年度 2009
  • 页码 164 p.
  • 总页数 164
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 化学;生物化学;
  • 关键词

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