A cost-effective colorimetric assay for phenolic O-methyltransferases and characterization of caffeate 3-O-methyltransferases from Populus trichocarpa.

Mohammad-Wadud Bhuiya, Chang-Jun Liu

Department of Biology, Brookhaven National Laboratory, Upton, NY 11973, USA.

Analytical biochemistry 2009 Jan 1

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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.

Citation

Mohammad-Wadud Bhuiya, Chang-Jun Liu. A cost-effective colorimetric assay for phenolic O-methyltransferases and characterization of caffeate 3-O-methyltransferases from Populus trichocarpa. Analytical biochemistry. 2009 Jan 1;384(1):151-8