A general fluorescence-based coupled assay for S-adenosylmethionine-dependent methyltransferases

We have developed a simple and sensitive fluorescence-based two-step coupled enzyme assay to report the activity of S-adenosylmethionine-dependent methyltransferases. This assay relies on a fluorescein-cystamine-methyl red (FL-S-S-MR) reporter molecule that can be activated by thiols. In the absence of thiols, fluorescence from the reporter is quenched through fluorescence resonance energy transfer between the two chromophores. In this report, we use catechol-O-methyltransferase with the addition of S-adenosylhomocysteine hydrolase to produce the thiol homocysteine. The presence of homocysteine leads to disulfide bond cleavage in the cystamine tether and fluorescence dequenching as the uncoupled chromophores are diluted into the surrounding media. The sensitivity and specificity of FL-S-S-MR to thiols enabled detection of 相似文献   

A liquid chromatography/mass spectrometry-based generic detection method for biochemical assay and hit discovery of histone methyltransferases

Epigenetic modifications of the genome, such as DNA methylation and posttranslational modifications of histone proteins, contribute to gene regulation. Growing evidence suggests that histone methyltransferases are associated with the development of various human diseases, including cancer, and are promising drug targets. High-quality generic assays will facilitate drug discovery efforts in this area. In this article, we present a liquid chromatography/mass spectrometry (LC/MS)-based S-adenosyl homocysteine (SAH) detection assay for histone methyltransferases (HMTs) and its applications in HMT drug discovery, including analyzing the activity of newly produced enzymes, developing and optimizing assays, performing focused compound library screens and orthogonal assays for hit confirmations, selectivity profiling against a panel of HMTs, and studying mode of action of select hits. This LC/MS-based generic assay has become a critical platform for our methyltransferase drug discovery efforts.     

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

A high-throughput, competitive fluorescence polarization immunoassay has been developed for the detection of methyltransferase activity. The assay was designed to detect S-adenosylhomocysteine (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 microM AdoMet. The limit of detection was approximately 5 nM (0.15 pmol) AdoHcy in the presence of 3 muM AdoMet. To validate the assay's ability to quantitate methyltransferase activity, the methyltransferase catechol-O-methyltransferase (COMT) and a known selective inhibitor of COMT activity were used in proof-of-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.     

A general liquid chromatography/mass spectroscopy-based assay for detection and quantitation of methyltransferase activity

Methyltransferases form a large class of enzymes, most of which use S-adenosylmethionine as the methyl donor. In fact, S-adenosylmethionine is second only to ATP in the variety of reactions for which it serves as a cofactor. Several methods to measure methyltransferase activity have been described, most of which are applicable only to specific enzymes and/or substrates. In this work we describe a sensitive liquid chromatography/mass spectroscopy-based methyltransferase assay. The assay monitors the conversion of S-adenosylmethionine to S-adenosylhomocysteine and can be applied to any methyltransferase and substrate of interest. We used the well-characterized enzyme catechol O-methyltransferase to demonstrate that the assay can monitor activity with a variety of substrates, can identify new substrates, and can be used even with crude preparation of enzyme. Furthermore, we demonstrate the utility of the assay for kinetic characterization of enzymatic activity.     

An enzyme-coupled continuous spectrophotometric assay for S-adenosylmethionine-dependent methyltransferases

Modification of small molecules and proteins by methyltransferases affects a wide range of biological processes. Here, we report an enzyme-coupled continuous spectrophotometric assay to quantitatively characterize S-adenosyl-L-methionine (AdoMet/SAM)-dependent methyltransferase activity. In this assay, S-adenosyl-L-homocysteine (AdoHcy/SAH), the transmethylation product of AdoMet-dependent methyltransferases, is hydrolyzed to S-ribosylhomocysteine and adenine by recombinant S-adenosylhomocysteine/5'-methylthioadenosine nucleosidase (SAHN/MTAN, EC 3.2.2.9). Subsequently, adenine generated from AdoHcy is further hydrolyzed to hypoxanthine and ammonia by recombinant adenine deaminase (EC 3.5.4.2). This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Coupling enzymes are recombinant and easily purified. The utility of this assay was shown using recombinant rat protein arginine N-methyltransferase 1 (PRMT1, EC 2.1.1.125), which catalyzes the mono- and dimethylation of guanidino nitrogens of arginine residues in select proteins. Using this assay, the kinetic parameters of PRMT1 with three synthetic peptides were determined. An advantage of this assay is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S-adenosylmethionine-dependent methyltransferases. Finally, this method may be used to assay other enzymes that produce AdoHcy, 5'-methylthioadenosine, or compounds that can be cleaved by AdoHcy nucleosidase.     

An enzyme-coupled continuous spectrophotometric assay for magnesium protoporphyrin IX methyltransferases

The second committed step in chlorophyll biosynthesis is the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to magnesium protoporphyrin IX (MgP) forming MgP monomethylester (MgPME). This reaction is catalyzed by the enzyme MgP methyltransferase (ChlM). Previous investigation of this enzyme has involved the use of time-consuming techniques requiring separation of products from substrates. More recent methyltransferase studies use coupling enzymes to monitor changes in absorption/fluorescence for the measurement of activity. However, due to the spectral properties of porphyrins, many of these assays are unsuitable for analysis of the catalytic properties of ChlM. Here we report the successful development of a coupled, continuous spectrophotometric assay to measure the activity of ChlM. The product of the methyltransferase reaction, S-adenosyl-l-homocysteine (SAH), is converted into adenine and then hypoxanthine by the recombinant coupling enzymes SAH nucleosidase and adenine deaminase, respectively. The appearance of hypoxanthine results in a decrease in absorbance at 265 nm.The utility of this assay was shown by the characterization of ChlM from the cyanobacterium Synechocystis sp. PCC 6803. Kinetic parameters obtained support data acquired using the discontinuous HPLC-based assay and provide further evidence for the stimulation of ChlM by the H subunit of magnesium chelatase (ChlH).     

An enzyme-coupled ultrasensitive luminescence assay for protein methyltransferases

Epigenetic regulation through protein posttranslational modifications is essential in development and disease. Among the key chemical modifications is protein methylation carried out by protein methyltransferases (PMTs). Quantitative and sensitive PMT activity assays can provide valuable tools to investigate PMT functions. Here we developed an enzyme-coupled luminescence assay for S-adenosyl-l-methionine (AdoMet/SAM)-based PMTs. In this assay, S-adenosyl-l-homocystine (AdoHcy/SAH), the by-product of PMT-involved methylation, is sequentially converted to adenine, adenosine monophosphate, and then adenosine 5′-triphosphate (ATP) by 5′-methylthio-adenosine/AdoHcy nucleosidase (MTAN), adenine phosphoribosyl transferase (APRT), and pyruvate orthophosphate dikinase (PPDK), respectively. The resultant ATP can be readily quantified with a luciferin/luciferase kit. This assay is featured for its quantitative linear response to AdoHcy and the ultrasensitivity to 0.3 pmol of AdoHcy. With this assay, the kinetic parameters of SET7/9 methylation were characterized and unambiguously support an ordered mechanism with AdoMet binding as the initial step, followed by the substrate binding and the rate-limiting methylation. The luminescence assay is also expected to be generally applicable to many other AdoMet-dependent enzymes. In addition, the mix-and-measure 96-/384-well format of our assay makes it suitable for automation and high throughput. Our enzyme-coupled luminescence assay, therefore, represents a convenient and ultrasensitive approach to examine methyltransferase activities and identify methyltransferase inhibitors.     

Histone lysine methyltransferases and demethylases in Plasmodium falciparum

Dynamic histone lysine methylation, regulated by methyltransferases and demethylases, plays fundamental roles in chromatin structure and gene expression in a wide range of eukaryotic organisms. A large number of SET-domain-containing proteins make up the histone lysine methyltransferase (HKMT) family, which catalyses the methylation of different lysine residues with relatively high substrate specificities. Another large family of Jumonji C (JmjC)-domain-containing histone lysine demethylases (JHDMs) reverses histone lysine methylation with both lysine site and methyl-state specificities. Through bioinformatic analysis, at least nine SET-domain-containing genes were found in the malaria parasite Plasmodium falciparum and its sibling species. Phylogenetic analysis separated these putative HKMTs into five subfamilies with different putative substrate specificities. Consistent with the phylogenetic subdivision, methyl marks were found on K4, K9 and K36 of histone H3 and K20 of histone H4 by site-specific methyl-lysine antibodies. In addition, most SET-domain genes and histone methyl-lysine marks displayed dynamic changes during the parasite asexual erythrocytic cycle, suggesting that they constitute an important epigenetic mechanism of gene regulation in malaria parasites. Furthermore, the malaria parasite and other apicomplexan genomes also encode JmjC-domain-containing proteins that may serve as histone lysine demethylases. Whereas prokaryotic expression of putative active domains of four P. falciparum SET proteins did not yield detectable HKMT activity towards recombinant P. falciparum histones, two protein domains expressed in vitro in a eukaryotic system showed HKMT activities towards H3 and H4, respectively. With the discovery of these Plasmodium SET- and JmjC-domain genes in the malaria parasite genomes, future efforts will be directed towards elucidation of their substrate specificities and functions in various cellular processes of the parasites.     

A spectrophotometric assay for measuring and detecting an epoxide hydrolase activity

In this paper we report the development of a novel and simple spectrophotometric assay which allows one to achieve the continuous, rapid, sensitive, and accurate determination of an epoxide hydrolase activity. This assay is based on the elaboration of a coupled enzymatic/chemical methodology which allows quantification of the enzymatic activity within 3min, and offers good sensitivity of about 10 micro Mmin(-1). Applicability of this test to some other aromatic epoxides has been shown and some limitations have also been explored. This assay should be particularly useful for different applications, for example (a) activity localization during purification of such enzymes, (b) very rapid determination of kinetic constants, and (c) high-throughput screening experiments.     

A continuous spectrophotometric enzyme-coupled assay for deoxynucleoside triphosphate triphosphohydrolases

We describe a continuous, spectrophotometric, enzyme-coupled assay useful to monitor reactions catalyzed by nucleoside triphosphohydrolases. In particular, using Escherichia coli deoxynucleoside triphosphohydrolase (Dgt), which hydrolyzes dGTP to deoxyguanosine and tripolyphosphate (PPP_i) as the enzyme to be tested, we devised a procedure relying on purine nucleoside phosphorylase (PNPase) and xanthine oxidase (XOD) as the auxiliary enzymes. The deoxyguanosine released by Dgt can indeed be conveniently subjected to phosphorolysis by PNPase, yielding deoxyribose-1-phosphate and guanine, which in turn can be oxidized to 8-oxoguanine by XOD. By this means, it was possible to continuously detect Dgt activity at 297 nm, at which wavelength the difference between the molar extinction coefficients of 8-oxoguanine (8000 M⁻¹ cm⁻¹) and guanine (1090 M⁻¹ cm⁻¹) is maximal. The initial velocities of Dgt-catalyzed reactions were then determined in parallel with the enzyme-coupled assay and with a discontinuous high-performance liquid chromatography (HPLC) method able to selectively detect deoxyguanosine. Under appropriate conditions of excess auxiliary enzymes, the activities determined with our continuous enzyme-coupled assay were quantitatively comparable to those observed with the HPLC method. Moreover, the enzyme-coupled assay proved to be more sensitive than the chromatographic procedure, permitting reliable detection of Dgt activity at low dGTP substrate concentrations.     

A fluorescent assay suitable for inhibitor screening and vanin tissue quantification

Vanin-1 is a pantetheinase that catalyzes the hydrolysis of pantetheine to produce pantothenic acid (vitamin B5) and cysteamine. Reported here is a highly sensitive fluorescent assay using a novel fluorescently labeled pantothenate derivative. The assay has been used for characterization of a soluble version of human vanin-1 recombinant protein, identification and characterization of hits from high-throughput screening (HTS), and quantification of vanin pantothenase activity in cell lines and tissues. Under optimized assay conditions, we quantified vanin pantothenase activity in tissue lysate and found low activity in lung and liver but high activity in kidney. We demonstrated that the purified recombinant vanin-1 consisting of the extracellular portion without the glycosylphosphatidylinositol (GPI) linker was highly active with an apparent K_m of 28 μM for pantothenate-7-amino-4-methylcoumarin (pantothenate-AMC), which was converted to pantothenic acid and AMC based on liquid chromatography-mass spectrometry (LC-MS) analysis. The assay also performed well in a 384-well microplate format under initial rate conditions (10% conversion) with a signal-to-background ratio (S/B) of 7 and a Z factor of 0.75. Preliminary screening of a library of 1280 pharmaceutically active compounds identified inhibitors with novel chemical scaffolds. This assay will be a powerful tool for target validation and drug lead identification and characterization.     

A stereospecific colorimetric assay for (S,S)-adenosylmethionine quantification based on thiopurine methyltransferase-catalyzed thiol methylation

S-Adenosyl-L-methionine (AdoMet) which is biologically synthesized by AdoMet synthetase bears an S configuration at the sulfur atom. The chiral sulfonium spontaneously racemizes to form a mixture of S and R isomers of AdoMet under physiological conditions or normal storage conditions. The chirality of AdoMet greatly affects its activity; the R isomer is not accepted as a substrate for AdoMet-dependent methyltransferases. We report a stereospecific colorimetric assay for (S,S)-adenosylmethionine quantification based on an enzyme-coupled reaction in which (S,S)-AdoMet reacts with 2-nitro-5-thiobenzoic acid to form AdoHcy and 2-nitro-5-methylthiobenzoic acid. The transformation is catalyzed by recombinant human thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) and is associated with a large spectral change at 410 nm. Accumulation of the S-adenosylhomocysteine (AdoHcy) product, a feedback inhibitor of TPMT, slows the assay. AdoHcy nucleosidase (EC 3.2.2.9) irreversibly cleaves AdoHcy to adenine and S-ribosylhomocysteine, significantly shortening the assay time to less than 10 min. The assay is linear from 5 to at least 60 microM (S,S)-AdoMet.     

Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC₅₀ for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors.     

A coupled assay measuring Mycobacterium tuberculosis antigen 85C enzymatic activity

The prevalence of drug-resistant strains of Mycobacterium tuberculosis (M. tb) emphasizes the need for new antitubercular drugs. An essential component of the drug discovery process is the development of tools to rapidly screen potential drug libraries against important biological targets. Similarly to well-documented M. tb targets, the antigen 85 (Ag85) enzymes are involved in the maintenance of the mycobacterial cell wall. The products synthesized by these mycolyltransferases are the cell wall components most responsible for the reduced permeability of drugs into the bacterial cell, thereby linking Ag85 activity directly with drug resistance. This article presents the development of a high-throughput colorimetric assay suitable for direct monitoring of the enzymatic activity. The assay uses a synthetic substrate containing three chemical moieties: an octanoyl fatty acid, β-d-glucose, and p-nitrophenyl. In the context of the assay, Ag85 catalyzes the removal of the fatty acid and releases p-nitrophenyl-β-d-glucoside. The glucoside is hydrolyzed by β-glucosidase to release the p-nitrophenolate chromophore. With this assay, the K_M and k_cat values of Ag85C were determined to be 0.047 ± 0.008 mM and 0.062 s⁻¹, respectively. In addition, the assay exhibits a Z′ value of 0.81 ± 0.06, indicating its suitability for high-throughput screening applications and drug development.     

Improved fluorogenic histone deacetylase assay for high-throughput-screening applications

Histone deacetylases (HDACs) are key targets for chemotherapeutic intervention in malignant diseases. In this paper, a highly sensitive, nonisotopic, homogeneous assay for high-throughput screening of HDAC inhibitors is presented. The assay is based on a new fluorogenic peptidic substrate of HDACs comprising an epsilon-acetylated lysyl moiety and an adjacent 4-methylcoumarin-7-amide moiety at the C terminus of the peptide chain. Upon deacetylation of the acetylated lysyl moiety, molecules are recognized as substrates by trypsin, which releases highly fluorescent 7-amino-4-methylcoumarin molecules in a subsequent step of the assay. The fluorescence increase is directly proportional to the amount of deacetylated substrate molecules, i.e., HDAC activity. Validation of an improved version of the assay revealed (i) a significantly lower enzyme consumption, (ii) an increased screening window coefficient, (iii) a good tolerance toward organic solvents, and (iv) a good suitability for a whole range of different HDAC-like enzymes. The novel assay thus will expedite studies of HDAC-like enzymes and in vitro screening for drug discovery.     

Two novel methyltransferases acting upon eukaryotic elongation factor 1A in Saccharomyces cerevisiae

Eukaryotic elongation factor 1A (eEF1A) is an abundant cytosolic protein in Saccharomyces cerevisiae and is well conserved amongst species. This protein undergoes multiple posttranslational modifications, including the N-methylation of four side chain lysine residues. However, the enzyme(s) responsible for catalyzing these modifications have remained elusive. Here we show by intact protein mass spectrometry that deletion of either of two genes coding for putative methyltransferases results in a loss in mass of eEF1A. Deletion of the YHL039W gene, a member of the SET domain subfamily including cytochrome c and ribosomal protein lysine methyltransferases, results in an eEF1A mass loss corresponding to a single methyl group. Deletion in the YIL064W/SEE1 gene, encoding a well conserved seven beta strand methyltransferase sequence, has been shown previously to affect vesicle transport; in this work we show that deletion results in the loss of two methyl group equivalents from eEF1A. We find that deletion of thirty-five other putative and established SET domain and seven beta strand methyltransferases has no effect on the mass of eEF1A. Finally, we show that wild type extracts, but not YIL064W/SEE1 mutant extracts, can catalyze the S-adenosylmethionine-dependent in vitro methylation of hypomethylated eEF1A. We suggest that YHL039W (now designated EFM1 for elongation factor methyltransferase 1) and YIL064W/SEE1 encode distinct eEF1A methyltransferases that respectively monomethylate and dimethylate this protein at lysine residues.     

Rapid homogeneous PCR assay for the detection of Chlamydia trachomatis in urine samples

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease and a major public health problem worldwide. Fast and sensitive point-of-care diagnostics including non-invasive sample collection would be of value for the prevention of C. trachomatis transmission. The aim of this study was to develop a fast, reliable, non-invasive and easy-to-use homogenous PCR assay for the detection of C. trachomatis. Bacteria were concentrated from urine by a simple and fast centrifugation-based urine pretreatment method. Novel automated GenomEra technology was utilized for the rapid closed-tube PCR including time-resolved fluorometric detection of the target using lanthanide chelate labeled probes. We have developed a rapid C. trachomatis assay which provides qualitative results in 1 h with diagnostic sensitivity and specificity of 98.7% and 97.3%, respectively. The novel assay can be performed with minimal laboratory expertise and without sophisticated DNA-extraction devices and has performance comparable to current gold standard assays.     

Structural snapshots of MTA/AdoHcy nucleosidase along the reaction coordinate provide insights into enzyme and nucleoside flexibility during catalysis

MTA/AdoHcy nucleosidase (MTAN) irreversibly hydrolyzes the N9-C1' bond in the nucleosides, 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (AdoHcy) to form adenine and the corresponding thioribose. MTAN plays a vital role in metabolic pathways involving methionine recycling, biological methylation, polyamine biosynthesis, and quorum sensing. Crystal structures of a wild-type (WT) MTAN complexed with glycerol, and mutant-enzyme and mutant-product complexes have been determined at 2.0A, 2.0A, and 2.1A resolution, respectively. The WT MTAN-glycerol structure provides a purine-free model and in combination with the previously solved thioribose-free MTAN-ADE structure, we now have separate apo structures for both MTAN binding subsites. The purine and thioribose-free states reveal an extensive enzyme-immobilized water network in their respective binding subsites. The Asp197Asn MTAN-MTA and Glu12Gln MTAN-MTR.ADE structures are the first enzyme-substrate and enzyme-product complexes reported for MTAN, respectively. These structures provide representative snapshots along the reaction coordinate and allow insight into the conformational changes of the enzyme and the nucleoside substrate. A "catalytic movie" detailing substrate binding, catalysis, and product release is presented.