Analysis of coenzyme A activated compounds in actinomycetes.
Analysis of coenzyme A activated compounds in actinomycetes.
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DOI：
10.1007/s00253-016-7635-0
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发表时间：
2016-08
影响因子：
5
通讯作者：
Gago G
中科院分区：
文献类型：
--
作者：
Cabruja M;Lyonnet BB;Millán G;Gramajo H;Gago G
酰基辅酶a是参与克雷布斯循环、脂质、碳水化合物和氨基酸代谢等重要代谢途径的关键化合物，也是许多生物体中参与脂质生物合成转录调控的关键信号分子。本研究利用质谱法的高选择性，建立了离子配对反相高压液相色谱电喷雾电离高分辨质谱法（IP-RP-HPLC/ESI-HRMS），对放线菌中广泛存在的脂肪酰基辅酶a进行了综合分析测定。使用QTOF光谱仪的优点在于在宽动态范围内具有优异的质量精度和可用于未知分析物分子式解析的真实同位素模式的测量。为了证明这一概念，我们使用该方法确定了分枝杆菌、链霉菌和棒状杆菌种中脂肪酰基coa池的组成，揭示了三个属之间以及两种所分析的分枝杆菌之间脂肪酰基coa的数量和种类分布的巨大差异；包括不同链长的羧基酰基辅酶a的存在，这是霉菌酸生物合成的关键底物。该方法还用于分析两种脂肪酸合成酶抑制剂对耻垢分枝杆菌酰基辅酶a谱的影响，结果显示异烟肼处理的细胞中出现了一些意想不到的低水平C24酰基辅酶a。这种稳健、灵敏、可靠的方法应广泛应用于酰基辅酶a分子参与的广泛细菌代谢的研究。
Acyl-CoAs are crucial compounds involved in essential metabolic pathways such as the Krebs cycle, lipid, carbohydrate and amino acid metabolism and they are also key signal molecules involved in the transcriptional regulation of lipid biosynthesis in many organisms. In this study we took advantage of the high selectivity of mass spectrometry and developed an ion-pairing reverse-phase high pressure liquid chromatography electrospray ionization high resolution mass spectrometry (IP-RP-HPLC/ESI-HRMS) method to carry on a comprehensive analytical determination of the wide range of fatty acyl-CoAs present in actinomycetes. The advantage of using a QTOF spectrometer resides in the excellent mass accuracy over a wide dynamic range and measurements of the true isotope pattern that can be used for molecular formula elucidation of unknown analytes. As a proof of concept we used this assay to determine the composition of the fatty acyl-CoA pools in Mycobacterium, Streptomyces and Corynebacterium species, revealing an extraordinary difference in fatty acyl-CoA amounts and species distribution between the three genera and between the two species of mycobacteria analyzed; including the presence of different chain-length carboxy-acyl-CoAs, key substrates of mycolic acid biosynthesis. The method was also used to analyze the impact of two fatty acid synthase inhibitors on the acyl-CoAs profile of Mycobacterium smegmatis which showed some unexpected low levels of C24 acyl-CoAs in the isoniazid treated cells. This robust, sensitive and reliable method should be broadly applicable in the studies of the wide range of bacteria metabolisms in which acyl-CoA molecules participate.