Submitted to: Methods in Enzymology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/28/2022
Publication Date: 11/23/2022
Citation: Sullivan, M.L. 2022. Preparation of hydroxycinnamoyl-coenzyme A thioesters using recombinant 4-coumarate:coenzyme A ligase (4CL) for characterization of BAHD hydroxycinnamoyltransferases enzyme activities. In: Jez, Joseph, editor. Methods in Enzymology. Volume 683. Cambridge, MA: Academic Press. p.3-18.
Interpretive Summary: BAHD hydroxycinnamoyl-coenzyme A (CoA) hydroxycinnamoyl transferases are a family of plant enzymes involved in making a number of compounds in plants, many of which play important roles in plant growth, development, and survival. These compounds also can have useful bioactivities with potential to be used as nutriceuticals. Biochemical characterization of these enzymes requires hydroxycinnamoyl-CoA thioesters as a reaction component, which can be difficult to make and expensive to purchase. Here, we described a simple procedure for making hydroxycinnamoyl-CoA thioesters enzymatically that is relatively inexpensive and does not require special skills or equipment. This information will be useful to researchers working on plant biochemical pathways that involve the BAHD hydroxycinnamoyl-CoA transferase family.
Technical Abstract: Analyses of the enzymatic activities of hydroxycinnamoyl-coenzyme A (CoA) hydroxycinnamoyl transferases require hydroxycinnamoyl-CoA thioesters as assay reagents. Here we describe an simple, cost effective method for preparing p-coumaroyl-, caffeoyl- and feruloyl-CoA thioesters using the Arabidopsis thaliana 4-coumarate:CoA ligase 1 (4CL1) expressed in Escherichia coli. Preparation of the 4CL enzyme, in vitro synthesis of the thioesters, and thioester purification utilizing a C-18 solid phase extraction column are detailed. The hydroxycinnamoyl-CoA thioesters produced are suitable for downstream qualitative and quantitative analyses.