A universal competitive fluorescence polarization activity assay for S-adenosylmethionine utilizing methyltransferases

Abstract

A high-throughput, competitive fluorescence polarization immunoassay has been developed for the detection of methyltransferase activity. The assay was designed to detect Sadenosylhomocysteine (AdoHcy), a product of all S-adenosylmethionine (AdoMet)-utilizing methyltransferase reactions. We employed commercially available anti-AdoHcy antibody and fluorescein-AdoHcy conjugate tracer to measure AdoHcy generated as a result of methyltransferase activity. AdoHcy competes with tracer in the antibody/tracer complex. The release of tracer results in a decrease in fluorescence polarization. Under optimized conditions, AdoHcy and AdoMet titrations demonstrated that the antibody had more than a 150-fold preference for binding AdoHcy relative to AdoMet. Mock methyltransferase reactions using both AdoHcy and AdoMet indicated that the assay tolerated 1 to 3 μM AdoMet. The limit of detection was approximately 5 nM (0.15 pmol) AdoHcy in the presence of 3 μM AdoMet. To validate the assay's ability to quantitate methyltransferase activity, the methyltransferase catechol-Omethyltransferase (COMT) and a known selective inhibitor of COMT activity were used in proofof-principle experiments. A time-and enzyme concentration-dependent decrease in fluorescence polarization was observed in the COMT assay that was developed. The IC 50 value obtained using a selective COMT inhibitor was consistent with previously published data. Thus, this sensitive and homogeneous assay is amenable for screening compounds for inhibitors of methyltransferase activity. KeywordsFluorescence polarization; Methyltransferase; S-Adenosylhomocysteine; S-Adenosylmethionine; Catechol-O-methyltransferase Methyltransferases are a diverse class of enzymes that catalyze the transfer of a single methyl group from a methyl donor molecule to a methyl acceptor. The vast majority use Sadenosylmethionine (AdoMet) 1 as the methyl donor [1,2]. AdoMet is the second most HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscriptwidely used substrate molecule in the cell after ATP. Methylation is an essential and highly choreographed regulatory metabolic process. Small organic molecules and peptides and most types of cellular macromolecules, including DNA, RNA, protein, and lipids, can serve as substrates for methylation (for detailed reviews, see Refs. [2][3][4][5][6][7]).A rapidly growing area of methyltransferase research focuses on methylation of histone tails by histone methyltransferases (HMTs) (for reviews, see Refs. [8,9]). Histone methylation is an epigenetic modification that has a role in formation of heterochromatin, X chromosome inactivation, and modification of gene expression patterns. Histone methylation occurs on arginine and lysine residues and is catalyzed by methyltransferases belonging to three distinct families of proteins: the protein arginine N-methyltransferase 1 (PRMT1) family, the SET-domain-containing protein family, and the non-SET-domain proteins DOT1/DOT1L. This histone methylation alters chromatin structure and, thus, regulates...

Direct Detection of Products from S-Adenosylmethionine-Dependent Enzymes Using a Competitive Fluorescence Polarization Assay

“…Interestingly, the LOD of AdoHcy for the developed competitive FP assay is notably better or comparable to previously published spectroscopic, fluorescence-based or FP immunoassays for measuring MTase activity. 14,15,23,34 Several developed assays for MTases require multiple coupled enzymes to manipulate generated products into producing an output signal, which can lower the sensitivity of the assay. Coupling the HpMTAN-D198N/AdoHcy-TAMRA complex directly to AdoMet-dependent enzymes provides a highly sensitive universal assay that immediately detects generated products without the complication of additional auxiliary enzymes.…”

Label-free electrochemical detection of human methyltransferase from tumors

“…The most abundant mammalian methyltransferase and an important diagnostic target is DNA(cytosine-5)-methyltransferase1 (DNMT1), which preferentially methylates hemimethylated DNA using the cofactor S-adenosyl-L-methionine (SAM) (7)(8)(9)(10). Current measurements of DNMT1 activity require [methyl-3 H]-SAM to observe radioactive labeling of DNA (8,11), or expensive fluorescence or colorimetric reagents with antibodies that require large instrumentation (12)(13)(14)(15), both of which are significant obstacles that impede more widespread assessment of DNMT1 activity.…”

Observing Biosynthetic Activity Utilizing Next Generation Sequencing and the DNA Linked Enzyme Coupled Assay

“…For AtIAMT1, the limited sequence counts for α-lipoic acid [9], phenol [10], N-acetylserotonin [16], xanthosine [17], 2-mercapto-5-nitrobenzimidazole [18], and 2-mercapto-5-nitrobenzimidazole [21] led us to consider these compounds as background activity. Considering all these facts, NGS-DLEnCA was able to confirm all previously reported substrates and identify five novel substrates for the three methyltransferases assayed.…”