Metabolomics approach for enzyme discovery
Metabolomics approach for enzyme discovery
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DOI：
10.1021/pr0600576
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发表时间：
2006-08-04
影响因子：
4.4
通讯作者：
Tomita, Masaru
中科院分区：
文献类型：
--
作者：
Saito, Natsumi;Robert, Martin;Tomita, Masaru
在功能基因组学中，寻找新的酶是一项重要而又困难的任务。在这里，我们提出了一种系统的方法，基于体外测定结合代谢物谱来发现新的酶活性。将复杂的代谢物混合物与纯化的候选蛋白质孵育，随后用毛细管电泳电喷雾电离质谱（CE-MS）对反应混合物进行分析。代谢物组成的特定变化可以直接表明酶活性的存在，而随后对其水平特异性变化的化合物的鉴定可以确定反应的实际底物和产物。我们首先使用几种大肠杆菌代谢酶对该方法进行了评估，然后将其应用于未表征蛋白的功能筛选。通过这种方式，发现YbhA和YbiV蛋白对不同的糖/磷酸糖具有磷酸转移酶和磷酸酶活性。我们的方法应该广泛适用于任何系统中的酶发现。
The search for novel enzymes is an important but difficult task in functional genomics. Here, we present a systematic method based on in vitro assays in combination with metabolite profiling to discover novel enzymatic activities. A complex mixture of metabolites is incubated with purified candidate proteins and the reaction mixture is subsequently profiled by capillary electrophoresis electrospray ionization mass spectrometry (CE-MS). Specific changes in the metabolite composition can directly suggest the presence of an enzymatic activity while subsequent identification of the compounds whose level changed specifically can pinpoint the actual substrate(s) and product(s) of the reaction. We first evaluated the method using several Escherichia coli metabolic enzymes and then applied it to the functional screening of uncharacterized proteins. In this manner, YbhA and YbiV proteins were found to display both phosphotransferase and phosphatase activity toward different sugars/sugar phosphates. Our approach should be broadly applicable and useful for enzyme discovery in any system.