Lethe Mary C L, Bui Dinh, Hu Ming, Wang Xiaoqiang, Singh Rashim, Chan Clement T Y
Department of Biomedical Engineering, College of Engineering, University of North Texas, 3940 N Elm Street, Denton, TX 76207, USA.
Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, 4349 Martin Luther King Boulevard, Houston, TX 77204, USA.
Int J Mol Sci. 2024 Feb 27;25(5):2725. doi: 10.3390/ijms25052725.
UDP-glycosyltransferases (UGTs) form a large enzyme family that is found in a wide range of organisms. These enzymes are known for accepting a wide variety of substrates, and they derivatize xenobiotics and metabolites for detoxification. However, most UGT homologs have not been well characterized, and their potential for biomedical and environmental applications is underexplored. In this work, we have used a fluorescent assay for screening substrates of a plant UGT homolog by monitoring the formation of UDP. We optimized the assay such that it could be used for high-throughput screening of substrates of the UGT enzyme, UGT71G1, and our results show that 34 of the 159 screened compound samples are potential substrates. With an LC-MS/MS method, we confirmed that three of these candidates indeed were glycosylated by UGT71G1, which includes bisphenol A (BPA) and 7-Ethyl-10-hydroxycamptothecin (SN-38); derivatization of these toxic compounds can lead to new environmental and medical applications. This work suggests that UGT homologs may recognize a substrate profile that is much broader than previously anticipated. Additionally, it demonstrates that this screening method provides a new means to study UDP-glycosyltransferases, facilitating the use of these enzymes to tackle a wide range of problems.
UDP-糖基转移酶（UGTs）构成了一个庞大的酶家族，广泛存在于各种生物体中。这些酶以能接受多种底物而闻名，它们通过衍生化异源生物和代谢物来实现解毒。然而，大多数UGT同源物尚未得到充分表征，其在生物医学和环境应用方面的潜力也未得到充分探索。在这项工作中，我们使用了一种荧光测定法，通过监测UDP的形成来筛选植物UGT同源物的底物。我们对该测定法进行了优化，使其可用于高通量筛选UGT酶UGT71G1的底物，我们的结果表明，在159个筛选的化合物样品中，有34个是潜在底物。通过液相色谱-串联质谱法（LC-MS/MS），我们证实其中三个候选物确实被UGT71G1糖基化，其中包括双酚A（BPA）和7-乙基-10-羟基喜树碱（SN-38）；这些有毒化合物的衍生化可带来新的环境和医学应用。这项工作表明，UGT同源物可能识别的底物谱比以前预期的要广泛得多。此外，它证明了这种筛选方法为研究UDP-糖基转移酶提供了一种新手段，有助于利用这些酶解决广泛的问题。
