title: Assays for S-adenosylmethionine (AdoMet/SAM)-dependent methyltransferases.
abstract: Modification of small molecules and proteins by methyltransferases impacts a wide range of biological processes. Here we report two methods for measuring methyltransferase activity. First we describe an enzyme-coupled continuous spectrophotometric assay used to quantitatively characterize S-adenosyl-L-methionine (AdoMet or SAM)-dependent methyltransferase activity. In this assay, S-adenosyl-L-homocysteine (AdoHcy or SAH), the transmethylation product of AdoMet-dependent methyltransferase, is hydrolyzed to S-ribohomocysteine and adenine by recombinant AdoHcy nucleosidase. Subsequently, the adenine generated from AdoHcy is further hydrolyzed to homoxanthine and ammonia by recombinant adenine deaminase. This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Secondly, we describe a discontinuous assay that follows radiolabel incorporation into the methyl receptor. An advantage of both assays is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S-adenosylmethionine-dependent methyltransferases. Importantly both methods are inexpensive, robust, and amenable to high throughput.
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University, Logan, Utah, USA.
authors.forename: Whitney L
authors.initials: WL
authors.lastname: Wooderchak
authors.forename: Zhaohui Sunny
authors.initials: ZS
authors.lastname: Zhou
authors.forename: Joan
authors.initials: J
authors.lastname: Hevel
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008780:Methyltransferases / Q000032:analysis* / Q000378:metabolism*; D015394:Molecular Structure; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
chemical_list: D012436:S-Adenosylmethionine; D008780:Methyltransferases