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Title:   Author  item
                                            Palmer, Nathan

                                                         - *Nate*item
                                            Sattler, Scottitem
                                            Saathoff, Aaronitem
                                            Sarath, Gautam
  Submitted to: Journal of Agricultural and Food Chemistry
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 3/24/2010
                                    Publication  Date: 4/19/2010
                                    Citation: Palmer, N.A., Sattler, S.E., Saathoff, A.J., Sarath, G. 2010. A Continuous, Quantitative Fluorescent Assay for Plant Caffeic acid O-Methyltransferases. Journal of Agricultural and Food Chemistry. 58: 5220-5226Interpretive Summary: Lignins are a major component of plant cell walls and represent a significant barrier for the efficient conversion of plant biomass into liquid fuels, such as ethanol.  Understanding enzymes and other plant factors controlling lignin biosynthesis in bioenergy species such as switchgrass and sorghum can be expected to assist in the breeding of improved germplasm.  In this study, a new enzymatic assay has been developed and validated to easily measure the activity of caffeic acid-O-methyltransferases (COMT) in plants. COMT is an important enzyme in the biogenesis of lignin in plants, and plants with low or no COMT activity have lower lignin and show improved conversion to ethanol.  This assay will therefore have utility in identifying switchgrass and sorghum plants with altered COMT activity, and can be used routinely within a breeding program to identify genotypes with potentially altered lignin content.
                                Technical Abstract: 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. (2005; Analytical Biochemistry 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 Km, and Vmax 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.


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