Spectrophotometric determination of reaction rates and kinetic parameters of a BAHD acyltransferase using DTNB (5,5'-dithio-bis-[2-nitrobenzoic acid])

Authors: Sullivan, Michael; BONAWITZ, NICHOLAS - Dow Agrosciences

Submitted to: Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/27/2018
Publication Date: 1/31/2018
Publication URL: http://handle.nal.usda.gov/10113/5922793
Citation: Sullivan, M.L., Bonawitz, N.D. 2018. Spectrophotometric determination of reaction rates and kinetic parameters of a BAHD acyltransferase using DTNB (5,5'-dithio-bis-[2-nitrobenzoic acid]). Plant Science. 269:148-152. doi: 10.1016/j.plantsci.2018.01.012
DOI: https://doi.org/10.1016/j.plantsci.2018.01.012

Interpretive Summary: In an effort to protect plants against biotic (such as insect and pathogen) and abiotic (such as ultraviolet light and ozone) stresses, researchers need to characterize the reactions of certain enzymes in the plant. One important group of enzymes, BAHD hydroxycinnamoyl-coenzyme A transferases, produces hydroxycinnamic acid compounds that play important roles in protecting plants and can act as dietary antioxidants. Characterizing the reactions of these enzymes can be laborious and time-consuming using the usual approach. This paper reports a fast and easy method of following hydroxycinnamoyl-coenzyme A transferase reactions by monitoring levels of coenzyme A. Because one molecule of coenzyme A is produced for every molecule of hydroxycinnamic acid derivative formed in the transferase reaction, coenzyme A can serve as a proxy for the hydroxycinnamic acid product. This method will be of interest to researchers working on this class of enzymes and should greatly facilitate characterization of these interesting and important enzymes. Better understanding of the underlying enzymatic reactions will allow understanding of how enzyme structure and function are related, and could allow enzymes with new functions to be created.

Technical Abstract: Hydroxycinnamoyl-Coenzyme A (CoA) hydroxycinnamoyl transferases are BAHD family acyltransferases that transfer hydroxycinnamoyl moieties from a CoA-thioester to an acceptor amine or alcohol to form an N-hydroxycinnamoyl amide or O-hydroxycinnamoyl ester, respectively, with the concomitant release of free CoA. One approach to measure reaction rates for these enzymes is to quantify the hydroxycinnamoyl amide or ester reaction product following chromatographic separation of reaction components. This approach can be labor-intensive and time-consuming. As an alternative, we examined the use of 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB, Ellman’s reagent) to spectrophotometrically quantify, in real time, the release of free CoA during the transferase reaction. Using a hydroxycinnamoyl-CoA:L-DOPA hydroxycinnamoyl transferase as a model, we show that DTNB has little to no effect on the transferase reaction and can be used to provide a good estimate of hydroxycinnamoyl amide formation, thus allowing for the quick and easy collection of reaction rate data and determination of transferase kinetic parameters. This approach should be applicable to a wide range of hydroxycinnamoyl-CoA and other BAHD acyltransferases.