Bhuiya Mohammad-Wadud, Liu Chang-Jun
Department of Biology, Brookhaven National Laboratory, Upton, NY 11973, USA.
Anal Biochem. 2009 Jan 1;384(1):151-8. doi: 10.1016/j.ab.2008.09.031. Epub 2008 Sep 25.
S-adenosyl-L-methionine (AdoMet)-dependent O-methyltransferases (OMTs) catalyze the transmethylation of a variety of phenolics in bacteria, plants, and humans. To rapidly characterize phenolic OMT activities, we adapted Gibbs' reagent, the dye originally used for detecting phenols, to develop a convenient assay method for measuring the catalytic properties of enzymatic transmethylation of phenolics. We demonstrated that Gibbs' reagent reacted with phenolics yielding distinct absorptive characters that we used to further develop the assay to monitor the reactivities of phenolic OMTs. To validate the method, we identified two caffeate/5-hydroxyferulate 3/5-O-methyltransferases (COMTs) from the black cottonwood, Populus trichocarpa. Together with a few other plant type I OMTs, we demonstrated that our Gibbs' reagent-mediated colorimetric assay could reliably determine the functions and kinetic parameters of phenolic OMTs. Because Gibbs' reagent reacting with different regioselectively modified phenolics displays different colorimetric properties, the assay method can be used to monitor both substrate specificity and the regioselectivity of phenolic OMTs.
S-腺苷-L-甲硫氨酸（AdoMet）依赖性O-甲基转移酶（OMTs）催化细菌、植物和人类中多种酚类的甲基转移反应。为了快速表征酚类OMT活性，我们对吉布斯试剂（最初用于检测酚类的染料）进行了改进，开发了一种方便的测定方法，用于测量酚类酶促甲基转移反应的催化特性。我们证明吉布斯试剂与酚类反应会产生独特的吸收特性，我们利用这些特性进一步改进了该测定方法，以监测酚类OMT的反应活性。为了验证该方法，我们从毛果杨中鉴定出了两种咖啡酸/5-羟基阿魏酸3/5-O-甲基转移酶（COMTs）。与其他一些植物I型OMT一起，我们证明了我们的吉布斯试剂介导的比色测定法能够可靠地确定酚类OMT的功能和动力学参数。由于与不同区域选择性修饰的酚类反应的吉布斯试剂表现出不同的比色特性，该测定方法可用于监测酚类OMT的底物特异性和区域选择性。
