Direct Detection of Products from S-Adenosylmethionine-Dependent Enzymes Using a Competitive Fluorescence Polarization Assay
Direct Detection of Products from S-Adenosylmethionine-Dependent Enzymes Using a Competitive Fluorescence Polarization Assay
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DOI：
10.1021/acs.analchem.7b03556
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发表时间：
2018-02-06
影响因子：
7.4
通讯作者：
Ronning, Donald R.
中科院分区：
文献类型：
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作者：
Banco, Michael T.;Mishra, Vidhi;Ronning, Donald R.
S-腺苷甲硫氨酸（S-Adenosylmethionine，MTMet）依赖性甲基转移酶（Methyltransferases，MTases）是一个重要的酶超家族，催化甲基转移到几个生物分子上。细胞组分甲基化的改变对重要的生物学过程产生关键影响，使MT酶成为治疗感染性疾病和由过度活跃的人类编码的MT酶引起的疾病的有吸引力的药物靶标。已经开发了几种方法用于监测MTase的活性，但大多数MTase测定具有固有的局限性或不适合高通量筛选。我们描述了一种通用的，竞争性的荧光偏振（FP）测定，直接测量生产的S-腺苷同型半胱氨酸（HCY）从MTases。我们开发的检测方法通过置换与催化惰性的5 '-甲基硫代腺苷核苷酶（MTAN-D198 N）变体复合的荧光标记的HCY分子来监测HCY的生成，该检测方法以混合和读取的形式进行。产生荧光标记的分子涉及通过将NH 4 Hcy与胺反应性罗丹明衍生物组合的一锅合成，该衍生物对MTAN-D198 N的Kd值为11.3 +/- 0.7 nM。所开发的竞争性FP测定法表达了6 nM的抗同型半胱氨酸的检测限，并且与抗同型半胱氨酸相比，对抗同型半胱氨酸表现出34倍的偏好。我们通过与NIH临床保藏中心进行中试筛选以及确定结核分枝杆菌EgtD的L-组氨酸甲基化动力学参数来证明所开发的检测方法的实用性。此外，所开发的检测方法通过检测各种含腺苷的分子（包括5 '-甲硫基腺苷、AMP和ADP），适用于其他蛋氨酸依赖性和ATP依赖性酶。
S-Adenosylmethionine (AdoMet)-dependent methyltransferases (MTases) are an essential superfamily of enzymes that catalyze the transfer of a methyl group to several biomolecules. Alterations in the methylation of cellular components crucially impact vital biological processes, making MTases attractive drug targets for treating infectious diseases and diseases caused by overactive human-encoded MTases. Several methods have been developed for monitoring the activity of MTases, but most MTase assays have inherent limitations or are not amenable for high-throughput screening. We describe a universal, competitive fluorescence polarization (FP) assay that directly measures the production of S-adenosylhomocysteine (AdoHcy) from MTases. Our developed assay monitors the generation of AdoHcy by displacing a fluorescently labeled AdoHcy molecule complexed to a catalytically inert 5'-methylthioadenosine nucleosidase (MTAN-D198N) variant performed in a mix-and-read format. Producing the fluorescently labeled molecule involves a one-pot synthesis by combining AdoHcy with an amine-reactive rhodamine derivative, which possesses a K-d value of 11.3 +/- 0.7 nM to MTAN-D198N. The developed competitive FP assay expresses a limit of detection for AdoHcy of 6 nM and exhibits a 34-fold preference to AdoHcy in comparison to AdoMet. We demonstrate the utility of the developed assay by performing a pilot screen with the NIH Clinical Collection as well as determining the kinetic parameters of L-histidine methylation for EgtD from Mycobacterium tuberculosis. Additionally, the developed assay is applicable to other AdoMet-dependent and ATP-dependent enzymes by detecting various adenosine-containing molecules including 5'-methylthioadenosine, AMP, and ADP.