A Simple and Efficient Method for the Substrate Identification of Amino Acid Decarboxylases.
A Simple and Efficient Method for the Substrate Identification of Amino Acid Decarboxylases.
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DOI：
10.3390/ijms232314551
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发表时间：
2022-11-22
影响因子：
5.6
通讯作者：
中科院分区：
文献类型：
--
作者：
氨基酸脱羧酶将氨基酸转化为不同的生物胺，调节各种生物过程。因此，鉴定氨基酸脱羧酶的底物对于研究脱羧酶的功能至关重要，特别是对于预测为氨基酸脱羧酶的新基因。本研究建立了一种基于LC-MS的氨基酸脱羧酶底物及酶活性鉴定方法。我们选择GAD 65和AADC作为模型来验证我们的方法。GAD 65和AADC在HEK 293 T细胞中表达并通过免疫沉淀纯化。纯化的氨基酸脱羧酶进行酶促反应与不同的底物混合物在体外。反应混合物的LC-MS分析鉴定了耗尽或积累的代谢物，其分别对应于候选酶底物和产物。我们的方法成功地鉴定了已知氨基酸脱羧酶的底物和产物。总之，我们的方法可以有效地鉴定氨基酸脱羧酶的底物和产物，这将有助于未来的氨基酸脱羧酶的研究。
Amino acid decarboxylases convert amino acids into different biogenic amines which regulate diverse biological processes. Therefore, identifying the substrates of amino acid decarboxylases is critical for investigating the function of the decarboxylases, especially for the new genes predicted to be amino acid decarboxylases. In the present work, we have established a simple and efficient method to identify the substrates and enzymatic activity of amino acid decarboxylases based on LC-MS methods. We chose GAD65 and AADC as models to validate our method. GAD65 and AADC were expressed in HEK 293T cells and purified through immunoprecipitation. The purified amino acid decarboxylases were subjected to enzymatic reaction with different substrate mixtures in vitro. LC-MS analysis of the reaction mixture identified depleted or accumulated metabolites, which corresponded to candidate enzyme substrates and products, respectively. Our method successfully identified the substrates and products of known amino acid decarboxylases. In summary, our method can efficiently identify the substrates and products of amino acid decarboxylases, which will facilitate future amino acid decarboxylase studies.
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影响因子：
2.9
作者：
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通讯作者：
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