A continuous, quantitative fluorescent assay for plant caffeic acid O-methyltransferases.
Plant caffeic acid O-methyltransferases (COMTs) use S-adenosylmethionine (ado-met), as a methyl donor to transmethylate their preferred (phenolic) substrates in vivo, and will generally utilize a range of phenolic compounds in vitro. Collazo et al. (Anal. Biochem. 2005, 342, 86-92) have published a discrete, end-point fluorescence assay to detect histone methyltransferases using S-adenosyl homocysteine hydrolase and adeonsine deaminase as coupling enzymes and a thiol-specific fluorophore, Thioglo1, as the detecting reagent. Using this previous assay as a guide, we have developed and validated a facile, sensitive and real-time fluorescence assay for characterizing plant COMTs and in the process simplified the original assay as well by obviating the need for adenosine deaminase in the assay, and simultaneously converting an end-point assay into a continuous one. Our assay has been used to kinetically characterize recombinant sorghum COMT (Bmr-12) a key enzyme involved in cell wall lignification, and analyze COMT activity in maturing tillers from switchgrass plants. Data indicated that the calculated K(m) and V(max) values for the recombinant sorghum COMT using different substrates in the fluorescent assay were similar to published values for COMT enzymes from other plant species. Native COMT activity was greatest in internodes at the top of a tiller and declined in the more basal internodes. This new assay should have broad applicability for characterizing COMTs and potentially other plant methlytransferases that utilize ado-met as a methyl donor.
Palmer
Nathan A
NA
Grain, Forage and Bioenergy Research Unit, USDA-ARS, Lincoln, Nebraska, USA.
Sattler
Scott E
SE
Saathoff
Aaron J
AJ
Sarath
Gautam
G
D001483:Base Sequence; D017931:DNA Primers; D008780:Methyltransferases / Q000032:analysis* / Q000235:genetics; D010944:Plants / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence / Q000379:methods*
D017931:DNA Primers; D008780:Methyltransferases; C019608:caffeate O-methyltransferase