Thapa Hem R, Lail Andrew J, Garg Neha, Agarwal Vinayak
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, United States.
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, United States; School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA, United States.
Methods Enzymol. 2018;604:333-366. doi: 10.1016/bs.mie.2018.01.028. Epub 2018 Mar 19.
Flavin-adenine dinucleotide (FAD)-dependent halogenases are widespread in natural product biosynthetic gene clusters and have been demonstrated to employ small organic molecules as substrates for halogenation, as well as substrates that are tethered to carrier proteins (CPs). Despite numerous reports of FAD-dependent halogenases utilizing CP-tethered substrates, only a few have been biochemically characterized due to limited accessibility to the physiological substrates. Here, we describe a method for the preparation of acyl-S-CP substrates and their use in biochemical assays to query the activity of FAD-dependent halogenases. Furthermore, we describe a mass spectrometry-based method for the characterization of acyl-S-CP substrates and the corresponding halogenated products generated by the halogenases. Finally, we test the substrate specificity of a physiological chlorinase and a physiological brominase from marine bacteria, and, for the first time, demonstrate the distinct halide specificity of halogenases. The methodology described here will enable characterization of new halogenases employing CP-tethered substrates.
黄素腺嘌呤二核苷酸（FAD）依赖性卤化酶广泛存在于天然产物生物合成基因簇中，并且已被证明能够利用小有机分子作为卤化底物，以及与载体蛋白（CP）相连的底物。尽管有大量关于FAD依赖性卤化酶利用与CP相连底物的报道，但由于生理底物的可及性有限，只有少数得到了生化表征。在此，我们描述了一种制备酰基-S-CP底物的方法及其在生化分析中用于探究FAD依赖性卤化酶活性的用途。此外，我们描述了一种基于质谱的方法，用于表征酰基-S-CP底物以及卤化酶产生的相应卤化产物。最后，我们测试了来自海洋细菌的一种生理叶绿素酶和一种生理溴化酶的底物特异性，并首次证明了卤化酶具有不同的卤化物特异性。本文所述方法将有助于表征采用与CP相连底物的新型卤化酶。
