title: An enzyme-coupled colorimetric assay for S-adenosylmethionine-dependent methyltransferases.
abstract: We report here an enzyme-coupled colorimetric assay for salicylic acid carboxyl methyltransferase (SAMT), which utilizes S-adenosyl-l-methionine (AdoMet or SAM) as the methyl donor. In this assay, S-adenosyl-l-homocysteine (AdoHcy or SAH), a common product of AdoMet-dependent transmethylation reactions, is first hydrolyzed by recombinant AdoHcy nucleosidase (EC 3.2.2.9) into adenine and S-ribosylhomocysteine. Recombinant LuxS (S-ribosylhomocysteinase, EC 3.2.1.148) cleaves the latter compound to form homocysteine. Finally, homocysteine is quantified using Ellman's reagent and the accompanying absorption change at 412nm through recording using a microplate format. Notably, SAMT and most AdoMet-dependent methyltransferases undergo marked AdoHcy-mediated product inhibition. As such, an additional advantage of this assay which includes AdoHcy nucleosidase is the destruction of AdoHcy, thus alleviating product inhibition. Under our assay conditions, complete substrate conversion is observed and precise kinetic parameters can be determined in a facile and quantitative manner. This assay should be generally applicable to other AdoMet-dependent methyltransferases. Moreover, the procedure is easily amendable to batch assay and high-throughput screening approaches.
authors.affiliation: Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
authors.forename: Cheryl L
authors.initials: CL
authors.lastname: Hendricks
authors.forename: Jeannine R
authors.initials: JR
authors.lastname: Ross
authors.forename: Eran
authors.initials: E
authors.lastname: Pichersky
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Noel
authors.forename: Zhaohui Sunny
authors.initials: ZS
authors.lastname: Zhou
mesh_terms: D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry / Q000295:instrumentation* / Q000379:methods*; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000032:analysis* / Q000378:metabolism*; D015394:Molecular Structure; D012435:S-Adenosylhomocysteine / Q000378:metabolism*
chemical_list: D012435:S-Adenosylhomocysteine; D008780:Methyltransferases; C110994:salicylic acid carboxyl methyltransferase