@article{32c99cfb0a0741738b4dd76a3280b0e1,
title = "A simplified characterization of: S -adenosyl-l-methionine-consuming enzymes with 1-Step EZ-MTase: A universal and straightforward coupled-assay for in vitro and in vivo setting",
abstract = "Methyltransferases use S-adenosyl-l-methionine (SAM) to deposit methyl marks. Many of these epigenetic 'writers' are associated with gene regulation. As cancer etiology is highly correlated with misregulated methylation patterns, methyltransferases are emerging therapeutic targets. Successful assignment of methyltransferases' roles within intricate biological networks relies on (1) the access to enzyme mechanistic insights and (2) the efficient screening of chemical probes against these targets. To characterize methyltransferases in vitro and in vivo, we report a highly-sensitive one-step deaminase-linked continuous assay where the S-adenosyl-l-homocysteine (SAH) enzyme-product is rapidly and quantitatively catabolized to S-inosyl-l-homocysteine (SIH). To highlight the broad capabilities of this assay, we established enzymatic characteristics of two protein arginine methyltransferases (PRMT5 and PRMT7), a histone-lysine N-methyltransferase (DIM-5) and a sarcosine/dimethylglycine N-methyltransferase (SDMT). Since the coupling deaminase TM0936 displays robust activity over a broad pH-range we determined the pH dependence of SDMT reaction rates. TM0936 reactions are monitored at 263 nm, so a drawback may arise when methyl acceptor substrates absorb within this UV-range. To overcome this limitation, we used an isosteric fluorescent SAM-analog: S-8-aza-adenosyl-l-methionine. Most enzymes tolerated this probe and sustained methyltransfers were efficiently monitored through loss of fluorescence at 360 nm. Unlike discontinuous radioactive- and antibody-based assays, our assay provides a simple, versatile and affordable approach towards the characterization of methyltransferases. Supported by three logs of linear dynamic range, the 1-Step EZ-MTase can detect methylation rates as low as 2 μM h-1, thus making it possible to quantify low nanomolar concentrations of glycine N-methyltransferase within crude biological samples. With Z′-factors above 0.75, this assay is well suited to high-throughput screening and may promote the identification of novel therapeutics.",
author = "Burgos, {Emmanuel S.} and Walters, {Ryan O.} and Huffman, {Derek M.} and David Shechter",
note = "Funding Information: We thank Dr Bruce R. Branchini (Connecticut College, New London, USA) for providing his original construct for expression of the wild-type Photinus pyralis luciferase (FLUC); Dr Vern L. Schramm (Albert Einstein College of Medicine) for sharing the pDEST14-AgAK, and pDEST14-SeMTAN vectors encoding the respective adenosine kinase from Anopheles gambiae, and S-adenosyl-L-homocysteine nucleosidase from Salmonella enterica; Dr Mar{\'i}a A. Pajares (Instituto de Investigaciones Bio-medicas “Alberto Sols”, Madrid, Spain) for forwarding the pET19b (pMj1208-1) vector encoding the His-tagged methionine adenosyltransferase from Methanococcus jannaschii; Dr Rui-Ming Xu (Institute of Biophysics, Chinese Academy of Sciences, Beijing, China) for his gi of the pET21a-CePRMT5; Dr Erik W. Debler (Laboratory of Cell Biology, The Rockefeller University, New-York, USA) for providing the pET28a-TbPRMT7 encoding wild-type PRMT7 from Trypanosoma brucei; Dr Erik U. Selker (Institute of Molecular Biology, University of Oregon, Eugene, USA) for the gi of pGST-DIM5 encoding wild-type histone H3 lysine MTase from Neurospora crassa (19-318; DIM-5); the DNASU Plasmid Repository for access to clones TmCD00084735 and GsCD00383580 for expression of deaminase TM0936 and GsSDMT, respectively; Dr Luka and Wagner (Vanderbilt University Medical Center, Nashville, USA) for their gi of pET-17b-HsGNMT vector encoding wild-type human glycine N-methyltransferase. Finally, we are thankful to Dr Sean Cahill and Edward Nieves (Albert Einstein College of Medicine) for their assistance in NMR and MS analyses. This work was supported by the American Cancer Society (Robbie Sue Mudd Kidney Cancer Research Scholar Grant 124891-RSG-13-396-01-DMC) and the National Institutes of Health (GM108646), both to David Shechter. Derek M. Huffman was supported by the National Institute on Aging (R00AG037574, R56AG052981 and P30AG038072), and the American Federation for Aging Research (AFAR). Ryan O. Walters was supported by a T32 training grant from the National Institute on Aging (T32AG23475). While the Bruker Avance IIIHD 300 MHz system from the Einstein Structural NMR Resource was purchased and is supported by the Albert Einstein College of Medicine, the Bruker Avance IIIHD 600 MHz system was purchased using funds from the National Institutes of Health (1S10OD016305).",
year = "2017",
doi = "10.1039/c7sc02830j",
language = "English (US)",
volume = "8",
pages = "6601--6612",
journal = "Chemical Science",
issn = "2041-6520",
publisher = "Royal Society of Chemistry",
number = "9",
}