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PubMed1999

Methylation pharmacogenetics: catechol O-methyltransferase, thiopurine methyltransferase, and histamine N-methyltransferase.

Methyl conjugation is an important pathway in the biotransformation of many exogenous and endogenous compounds. Pharmacogenetic studies of methyltransferase enzymes have resulted in the identification and characterization of functionally important common genetic polymorphisms for catechol O-methyltransferase, thiopurine methyltransferase, and histamine N-methyltransferase. In recent years, characterization of these genetic polymorphisms has been extended to include the cloning of cDNAs and genes, as well as a determination of the molecular basis for the effects of inheritance on these methyltransferase enzymes. The thiopurine methyltransferase genetic polymorphism is responsible for clinically significant individual variations in the toxicity and therapeutic efficacy of thiopurine drugs such as 6-mercaptopurine. Phenotyping for the thiopurine methyltransferase genetic polymorphism represents one of the first examples in which testing for a pharmacogenetic variant has entered standard clinical practice. The full functional implications of pharmacogenetic variation in the activities of catechol O-methyltransferase and histamine N-methyltransferase remain to be determined. Finally, experimental strategies used to study methylation pharmacogenetics illustrate the rapid evolution of biochemical, pharmacologic, molecular, and genomic approaches that have been used to determine the role of inheritance in variation in drug metabolism, effect, and toxicity.

Animals↗
PubMed1993

S-methyltransferases in human intestine: differential distribution of the microsomal thiol methyltransferase and cytosolic thiopurine methyltransferase along the human bowel.

1. The activities of the microsomal thiol methyltransferase and the cytosolic thiopurine methyltransferase were measured with 2-mercaptoethanol and 6-mercaptopurine as substrates in human ileum, ascending colon, transverse colon, descending colon and liver. 2. Thiol methyltransferase activity (pmol/min per mg) (mean +/- SD) was 495 +/- 280 (ileum), 786 +/- 454 (ascending colon), 1791 +/- 233 (transverse colon), 964 +/- 484 (descending colon) and 4800 +/- 1194 (liver). 3. Thiopurine methyltransferase (pmol/min per mg) (mean +/- SD) was 53.5 +/- 15.4 (ileum), 34.6 +/- 11.4 (ascending colon), 64.3 +/- 12.1 (transverse colon), 57.0 +/- 10.1 (descending colon) and 106 +/- 20.4 (liver). 4. Transferase in intestinal mucosa followed non-Michaelis-Menten kinetics, and two phases representing high and low affinity forms, for the acceptor methyl substrates were observed. 5. Comparison of intestinal with hepatic activities showed that thiopurine methyltransferase is better expressed than thiol methyltransferase in the human intestine, at least with the substrates studied.

Adult↗
PubMed1996

Involvement of methyltransferase-activating protein and methyltransferase 2 isoenzyme II in methylamine:coenzyme M methyltransferase reactions in Methanosarcina barkeri Fusaro.

The enzyme systems involved in the methyl group transfer from methanol and from tri- and dimethylamine to 2-mercaptoethanesulfonic acid (coenzyme M) were resolved from cell extracts of Methanosarcina barkeri Fusaro grown on methanol and trimethylamine, respectively. Resolution was accomplished by ammonium sulfate fractionation, anion-exchange chromatography, and fast protein liquid chromatography. The methyl group transfer reactions from tri- and dimethylamine, as well as the monomethylamine:coenzyme M methyltransferase reaction, were strictly dependent on catalytic amounts of ATP and on a protein present in the 65% ammonium sulfate supernatant. The latter could be replaced by methyltransferase-activating protein isolated from methanol-grown cells of the organism. In addition, the tri- and dimethylamine:coenzyme M methyltransferase reactions required the presence of a methylcobalamin:coenzyme M methyltransferase (MT2), which is different from the analogous enzyme from methanol-grown M. barkeri. In this work, it is shown that the various methylamine:coenzyme M methyltransfer steps proceed in a fashion which is mechanistically similar to the methanol:coenzyme M methyl transfer, yet with the participation of specific corrinoid enzymes and a specific MT2 isoenzyme.

Adenosine Triphosphate↗
PubMed1980

Hydroxyindole-O-methyltransferase (HIOMT), catechol-O-methyltransferase (COMT) and histamine-N-methyltransferase (HNMT) in the pineal gland of the vizcacha (Lagostomus maximus maximus).

The activities of three methylating enzymes HIOMT, COMT, and HNMT were determined in pineal glands from 2 groups of adult vizcachas of both sexes, one (I) maintained under permanent lighting (15 days) and the other (II) kept in darkness. The control determinations (III) were carried out in pineal glands from animals hunted and killed during the night. In (I) the HIOMT activity decreased and the COMT activity increased; no changes were exhibited by HNMT. In (II) showed a diminished HIOMT activity. In captivity illumination affected HIOMT and COMT activites in a similar form to that observed in other species of rodents. The decrease in HIOMT activity in (II) suggests that captivity may affect pineal function, possibly as a consequence of a decrease in locomotor activity.

Acetylserotonin O-Methyltransferase↗
PubMed2001

Arylamine N-methyltransferase and thiol methyltransferase activities in cholestatic rat liver induced by common bile duct ligation.

Methylation catalyzed by methyltransferases is a major metabolic pathway for an inactivation of some catecholamines, niacinamide as well as aliphatic sulfhydryl drugs and toxic hydrogen sulfides. To investigate the effects of obstructive jaundice in an animal model, common bile duct ligation (CBDL) was performed in the rat and enzyme activities of S-adenosyl-L-methionine-dependent arylamine N-methyltransferase and thiol methyltransferase were examined in liver cell fractions and serum for a period of 42 d after CBDL. Both mitochondrial and microsomal arylamine N-methyltransferase showed significant increases in their activities between the 1st through the 7th day (P < or = 0.05 to 0.001), and between the 1st through the 28th day (P < or = 0.01 to 0.001) post-ligation, although the cytosolic arylamine N-methyltransferase activity did not show a significant change compared to the activities from the sham-operated control. The mitochondrial as well as microsomal thiol methyltransferase showed significant increases in their activities between the 1st through the 28th day (P < or = 0.05 to 0.01 and P < or = 0.01 to 0.001, respectively) post-ligation, although the cytosolic thiol methyltransferase activity did not show a significant change compared to the activities from the sham-operated control. Arylamine N-methyltransferase and thiol methyltransferase in the serum from cholestatic rats also showed significant increases in their activities between the 1st through 28th day (P < or = 0.01 to 0.001), and between the 0.5th through the 42nd day (P < or = 0.05 to 0.001) post-ligation compared to the sham-operated control, respectively. Enzyme kinetic parameters (Km and Vmax) of hepatic membrane-bound arylamine N-methyltransferase and thiol methyltransferase were analyzed with the preparation from the 7th day post-ligation, using tryptamine or 4-chlorothiophenol as substrates and S-Adenosyl-L-[methyl-3H]methionine as co-substrate. The results indicate that although the Km values were about the same as the sham-operated control, the Vmax values of both enzymes increased significantly (P < or = 0.01 and 0.001, respectively). These results suggest that the biosynthesis of arylamine N-methyltransferase and thiol methyltransferase have been induced in response to obstructive jaundice.

Animals↗
PubMed2002

Phenylethanolamine N-methyltransferase has beta-carboline 2N-methyltransferase activity: hypothetical relevance to Parkinson's disease.

Mammalian brain has a beta-carboline 2N-methyltransferase activity that converts beta-carbolines, such as norharman and harman, into 2N-methylated beta-carbolinium cations, which are structural and functional analogs of the Parkinsonian-inducing toxin 1-methyl-4-phenylpyridinium cation (MPP+). The identity and physiological function of this beta-carboline 2N-methylation activity was previously unknown. We report pharmacological and biochemical evidence that phenylethanolamine N-methyltransferase (EC 2.1.1.28) has beta-carboline 2N-methyltransferase activity. Specifically, purified phenylethanolamine N-methyltransferase (PNMT) catalyzes the 2N-methylation (21.1 pmol/h per unit PNMT) of 9-methylnorharman, but not the 9N-methylation of 2-methylnorharmanium cation. LY134046, a selective inhibitor of phenylethanolamine N-methyltransferase, inhibits (IC50 1.9 microM) the 2N-methylation of 9-methylnorharman, a substrate for beta-carboline 2N-methyltransferase. Substrates of phenylethanolamine N-methyltransferase also inhibit beta-carboline 2N-methyltransferase activity in a concentration-dependent manner. beta-Carboline 2N-methyltransferase activity (43.7pmol/h/mg protein) is present in human adrenal medulla, a tissue with high phenylethanolamine N-methyltransferase activity. We are investigating the potential role of N-methylated beta-carbolinium cations in the pathogenesis of idiopathic Parkinson's disease. Presuming that phenylethanolamine N-methyltransferase activity forms toxic 2N-methylated beta-carbolinium cations, we propose a novel hypothesis regarding Parkinson's disease-a hypothesis that includes a role for phenylethanolamine N-methyltransferase-catalyzed formation of MPP+ -like 2N-methylated beta-carbolinium cations.

Adrenal Medulla↗
PubMed2000

The trimethylamine methyltransferase gene and multiple dimethylamine methyltransferase genes of Methanosarcina barkeri contain in-frame and read-through amber codons.

Three different methyltransferases initiate methanogenesis from trimethylamine (TMA), dimethylamine (DMA) or monomethylamine (MMA) by methylating different cognate corrinoid proteins that are subsequently used to methylate coenzyme M (CoM). Here, genes encoding the DMA and TMA methyltransferases are characterized for the first time. A single copy of mttB, the TMA methyltransferase gene, was cotranscribed with a copy of the DMA methyltransferase gene, mtbB1. However, two other nearly identical copies of mtbB1, designated mtbB2 and mtbB3, were also found in the genome. A 6.8-kb transcript was detected with probes to mttB and mtbB1, as well as to mtbC and mttC, encoding the cognate corrinoid proteins for DMA:CoM and TMA:CoM methyl transfer, respectively, and with probes to mttP, encoding a putative membrane protein which might function as a methylamine permease. These results indicate that these genes, found on the chromosome in the order mtbC, mttB, mttC, mttP, and mtbB1, form a single transcriptional unit. A transcriptional start site was detected 303 or 304 bp upstream of the translational start of mtbC. The MMA, DMA, and TMA methyltransferases are not homologs; however, like the MMA methyltransferase gene, the genes encoding the DMA and TMA methyltransferases each contain a single in-frame amber codon. Each of the three DMA methyltransferase gene copies from Methanosarcina barkeri contained an amber codon at the same position, followed by a downstream UAA or UGA codon. The C-terminal residues of DMA methyltransferase purified from TMA-grown cells matched the residues predicted for the gene products of mtbB1, mtbB2, or mtbB3 if termination occurred at the UAA or UGA codon rather than the in-frame amber codon. The mttB gene from Methanosarcina thermophila contained a UAG codon at the same position as the M. barkeri mttB gene. The UAG codon is also present in mttB transcripts. Thus, the genes encoding the three types of methyltransferases that initiate methanogenesis from methylamine contain in-frame amber codons that are suppressed during expression of the characterized methyltransferases.

Amino Acid Sequence↗
PubMed1995

Enzymatic methylation of arsenic compounds: assay, partial purification, and properties of arsenite methyltransferase and monomethylarsonic acid methyltransferase of rabbit liver.

A rapid, accurate, in vitro assay utilizing radioactive S-adenosylmethionine (SAM) has been developed for the methylation of arsenite and monomethylarsonate (MMA) by rabbit liver methyltransferases. The assay has been validated by separating, identifying, and measuring the products of the reaction using chloroform extraction, ion exchange chromatography, TLC, or HPLC. The enzymes involved in this pathway, arsenite methyltransferase and MMA methyltransferase, have been purified approximately 2000-fold from rabbit liver. After gel electrophoresis, a single band is obtained with both enzyme activities in it. The pH optima for purified arsenite methyltransferase and monomethylarsonic acid methyltransferase are 8.2 and 8.0, respectively. A thiol, S-adenosylmethionine, and arsenite are required for the partially purified arsenite methyltransferase that catalyzes the synthesis of monomethylarsonate. A different enzyme activity that catalyzes the methylation of monomethylarsonate to dimethylarsinate also requires SAM and a thiol. Even though arsenite methyltransferase and monomethylarsonate methyltransferase have different substrates, pH optima, and saturation concentrations for their substrates, whether the two activities are present on one protein molecule or different protein molecules is still uncertain. Both activities have a molecular mass of 60 kDa as determined by gel exclusion chromatography. There is no evidence at the present time for these enzyme activities being on different protein molecules. Neither arsenate, selenate, selenite, or selenide are methylated by the purified enzyme preparations. Results from the use of crude extracts, often called cytosol, to study the properties of these methyltransferases dealing with arsenic species should be viewed with caution since such crude extracts contain inhibiting and other interfering activities.

Animals↗
PubMed1984

The effect of hyper and hypothyroidism, hypophysectomy and adrenalectomy on phosphatidylethanolamine methyltransferase, phosphatidyldimethyl-ethanolamine methyltransferase and choline phosphotransferase of rat liver microsomes.

The effect of hyper- and hypothyroid, hypophysectomy and adrenalectomy on phosphatidylcholine biosynthetic enzymes, phosphatidylethanolamine methyltransferase, phosphatidyldimethylethanolamine methyltransferase and choline phosphotransferase of liver microsomes was measured in rats. There was a significant increase in the specific activity of phosphatidylethanolamine methyltransferase in the hyperthyroid rats. There was a significant reduction in the specific activity of phosphatidylethanolamine methyltransferase and phosphatidyldimethylethanolamine methyltransferase in the hypothyroid states. The choline phosphotransferase increased significantly in the hyperthyroid state and decreased in the hypothyroid animals. Hypophysectomy resulted in a significant increase in specific activity of choline phosphotransferase. A reduction in the specific activity of the phosphatidylethanolamine methyltransferase occurred after 28 days of hypophysectomy. Adrenalectomy resulted in a significant stimulation of the specific activity of phosphatidylethanolamine methyltransferase and choline phosphotransferase in liver microsomes.

Adrenalectomy↗
PubMed1999

On the substrate specificity of DNA methyltransferases. adenine-N6 DNA methyltransferases also modify cytosine residues at position N4.

Methylation of DNA is important in many organisms and essential in mammals. Nucleobases can be methylated at the adenine-N6, cytosine-N4, or cytosine-C5 atoms by specific DNA methyltransferases. We show here that the M.EcoRV, M.EcoRI, and Escherichia coli dam methyltransferases as well as the N- and C-terminal domains of the M. FokI enzyme, which were formerly all classified as adenine-N6 DNA methyltransferases, also methylate cytosine residues at position N4. Kinetic analyses demonstrate that the rate of methylation of cytosine residues by M.EcoRV and the M.FokI enzymes is reduced by only 1-2 orders of magnitude in relation to methylation of adenines. This result shows that although these enzymes methylate DNA in a sequence specific manner, they have a low substrate specificity with respect to the target base. This unexpected finding has implications on the mechanism of adenine-N6 DNA methyltransferases. Sequence comparisons suggest that adenine-N6 and cytosine-N4 methyltransferases have changed their reaction specificity at least twice during evolution, a model that becomes much more likely given the partial functional overlap of both enzyme types. In contrast, methylation of adenine residues by the cytosine-N4 methyltransferase M.BamHI was not detectable. On the basis of our results, we suggest that adenine-N6 and cytosine-N4 methyltransferases should be grouped into one enzyme family.

Adenine↗
PubMed2000

Structure of RsrI methyltransferase, a member of the N6-adenine beta class of DNA methyltransferases.

DNA methylation is important in cellular, developmental and disease processes, as well as in bacterial restriction-modification systems. Methylation of DNA at the amino groups of cytosine and adenine is a common mode of protection against restriction endonucleases afforded by the bacterial methyltransferases. The first structure of an N:6-adenine methyltransferase belonging to the beta class of bacterial methyltransferases is described here. The structure of M. RSR:I from Rhodobacter sphaeroides, which methylates the second adenine of the GAATTC sequence, was determined to 1.75 A resolution using X-ray crystallography. Like other methyltransferases, the enzyme contains the methylase fold and has well-defined substrate binding pockets. The catalytic core most closely resembles the PVU:II methyltransferase, a cytosine amino methyltransferase of the same beta group. The larger nucleotide binding pocket observed in M. RSR:I is expected because it methylates adenine. However, the most striking difference between the RSR:I methyltransferase and the other bacterial enzymes is the structure of the putative DNA target recognition domain, which is formed in part by two helices on an extended arm of the protein on the face of the enzyme opposite the active site. This observation suggests that a dramatic conformational change or oligomerization may take place during DNA binding and methylation.

Adenine↗
PubMed1993

Diethyldithiocarbamate S-methylation: evidence for catalysis by human liver thiol methyltransferase and thiopurine methyltransferase.

Disulfiram is used in the treatment of alcoholism to inhibit the enzyme aldehyde dehydrogenase. Disulfiram is rapidly reduced in vivo to form diethyldithiocarbamate (DDC), and DDC can undergo methyl conjugation to form S-methyl-DDC. Human tissues contain two separate genetically regulated enzymes that can catalyze thiol S-methylation. Thiol methyltransferase (TMT) is a microsomal enzyme that preferentially catalyzes, the S-methylation of alipathic sulfhydryl compounds, whereas thiopurine methyltransferase (TPMT) is a cytoplasmic enzyme that preferentially catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds. Our experiments were performed to determine whether human liver microsomal and/or cytosolic preparations could catalyze the S-methylation of DDC, and, if so, to determine whether TMT or TPMT might be the enzymes involved. We found that both human liver microsomes and cytosol could catalyze DDC S-methylation. The microsomal activity displayed biphasic substrate kinetics, with apparent Km values for DDC of 7.9 and 1500 microM for the high- and low-affinity activities, respectively. The high-affinity activity had an apparent Km value for S-adenosyl-L-methionine, the methyl donor for the reaction, of 5.8 microM. The thermal inactivation profile and response to methyltransferase inhibitors of the high-affinity microsomal DDC S-methyltransferase activity were similar to those of human liver microsomal TMT. In addition, TMT activity and the activity catalyzing the S-methylation of DDC were highly correlated in 19 individual liver samples (rs = 0.956; P < .0001). Hepatic cytosolic DDC S-methyltransferase activity had an apparent Km value for DDC of 95 microM. The cytosolic enzyme which catalyzed DDC S-methylation and TPMT activity had similar thermal inactivation profiles, similar patterns of response to methyltransferase inhibitors and the two activities coeluted during ion exchange chromatography. Furthermore, the activities of TPMT and cytosolic DDC S-methyltransferase were highly correlated in 20 individual liver samples (rs = 0.963; P < .0001). These results were compatible with the conclusion that both TMT and TPMT could catalyze the S-methylation of DDC in the human liver. Because the activities of both TMT and TPMT are controlled by inheritance, our observations raise the possibility of pharmacogenetic variation in the biotransformation and therapeutic effect of DDC in humans.

Aldehyde Dehydrogenase↗
PubMed2006

The histone methyltransferase SETDB1 and the DNA methyltransferase DNMT3A interact directly and localize to promoters silenced in cancer cells.

DNA CpG methylation can cooperate with histone H3 lysine 9 (H3-K9) methylation in heterochromatin formation and gene silencing. Trimethylation of H3-K9 by the recently identified euchromatic histone methyltransferase SETDB1/ESET may be responsible for transcriptional repression of certain promoters. Here, we show that SETDB1 associates with endogenous DNA methyltransferase activity. SETDB1 interacts with the de novo DNA methyltransferases DNMT3A and DNMT3B but not with the maintenance methyltransferase DNMT1. The interaction of SETDB1 with DNMT3A was further characterized and confirmed by in vivo and in vitro interaction studies. A direct interaction of the two proteins occurs through the N terminus of SETDB1 and the plant homeodomain of DNMT3A. Co-expression of SETDB1 and DNMT3A was essential for repression of reporter gene expression in a Gal4-based tethering assay and resulted in their recruitment to the artificial promoter. We further demonstrate that the CpG-methylated promoters of the endogenous p53BP2 gene in HeLa cells and the RASSF1A gene in MDA-MB-231 cells are simultaneously occupied by both SETDB1 and DNMT3A proteins, which provides evidence for SETDB1 being at least partly responsible for H3-K9 trimethylation at the promoter of RASSF1A, a gene frequently silenced in human cancers. In summary, our data demonstrate the direct physical interaction and functional connection between the H3-K9 trimethylase SETDB1 and the DNA methyltransferase DNMT3A and thus contribute to a better understanding of the complexity of the self-reinforcing heterochromatin machinery operating at silenced promoters.

Animals↗
PubMed2003

The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase.

The DNA methyltransferases, Dnmts, are the enzymes responsible for methylating DNA in mammals, which leads to gene silencing. Repression by DNA methylation is mediated partly by recruitment of the methyl-CpG-binding protein MeCP2. Recently, MeCP2 was shown to associate and facilitate histone methylation at Lys9 of H3, which is a key epigenetic modification involved in gene silencing. Here, we show that endogenous Dnmt3a associates primarily with histone H3-K9 methyltransferase activity as well as, to a lesser extent, with H3-K4 enzymatic activity. The association with enzymatic activity is mediated by the conserved PHD-like motif of Dnmt3a. The H3-K9 histone methyltransferase that binds Dnmt3a is likely the H3-K9 specific SUV39H1 enzyme since we find that it interacts both in vitro and in vivo with Dnmt3a, using its PHD-like motif. We find that SUV39H1 also binds to Dnmt1 and, consistent with these interactions, SUV39H1 can purify DNA methyltransferase activity from nuclear extracts. In addition, we show that HP1beta, a SUV39H1-interacting partner, binds directly to Dnmt1 and Dnmt3a and that native HP1beta associates with DNA methyltransferase activity. Our data show a direct connection between the enzymes responsible for DNA methylation and histone methylation. These results further substantiate the notion of a self-reinforcing repressive chromatin state through the interplay between these two global epigenetic modifications.

Blotting, Western↗
PubMed1983

Correlation of low and high affinity thiol methyltransferase and phenol methyltransferase activities in human erythrocyte membranes.

Human red blood cell (RBC) membranes have been reported to contain both high and low affinity 'forms' of the drug metabolizing enzyme thiol methyltransferase (TMT). The biochemical characteristics of the two 'forms' of human RBC TMT were compared. Apparent Km constants of the high affinity activity for 2-mercaptoethanol and S-adenosyl-L-methionine, cosubstrates for the TMT reaction, were 0.38 mumol/l and 2.6 mumol/l, respectively. These constants may be compared with values of 20 mmol/l and 43 mumol/l, respectively, previously reported for the low affinity form of RBC TMT activity. The properties and regulation of the two forms of TMT were then compared with each other and also with those of two 'control' enzymes, phenol methyltransferase (PMT) and beta-glucuronidase. When high and low affinity TMT, PMT and beta-glucuronidase activities were measured in RBC membranes from 22 individual subjects, there were highly significant correlations among all three methyltransferase activities (all r values greater than 0.95), but beta-glucuronidase activity did not correlate significantly with any of the methyltransferase activities (all r values less than 0.40). The thermal stabilities of the three methyltransferases were very similar. They were all inactivated approximately 50% by incubation at 48 degrees C for 15 min. beta-Glucuronidase activity was approximately 50% inactivated by incubation at 76 degrees C for 15 min. PMT and both TMT activities had similar subcellular distributions and similar responses to ions and to enzyme inhibitors. These results suggested that high and low affinity TMT and PMT activities might be catalyzed by the same enzyme. Alternatively, these three RBC membrane methyltransferase activities might be regulated in a parallel fashion.

Adult↗
PubMed1979

Affinity chromatography of Ruta graveolens L. O-methyltransferases. Studies demonstrating the potential of the technique in the mechanistic investigation of O-methyltransferases.

Two discrete furanocoumarin (5- and 8-)O-methyltransferases and a caffeic acid 3-O-methyl-transferase from cell cultures of Ruta graveoleus L. have been copurified by affinity chromatography on 1,6-diaminohexane agarose (AH-Sepharose 4B) linked with S-adenosyl-L-homocysteine (SAH). The furanocoumarin O-methyltransferases, which transfer a methyl group from S-adenosyl-L-methionine (SAM) to the 5- or 8-hydroxyls of linear furanocoumarins, were not retarded by 5-(3-carboxypropanamido)-xanthotoxin (CPAX) immobilized to AH-Sepharose 4B, but addition of SAM to the irrigant buffer led to complete retardation of both enzymes on this affinity system. An analogous phenomenon was observed for the caffeic acid O-methyltransferase, with a ferulic acid ligand coupled to the same insoluble support. SAH was as effective as SAM in promoting binding of the furanocoumarin O-methyltransferases to CPAX and caffeic acid 3-O-methyltransferase to immobilized ferulic acid, respectively. The strong and specific adsorption of these enzymes was abolished by exclusion of SAM or SAH from the irrigant buffer. It is concluded that the enzymes bind first to SAM or SAH, and that this binding process in turn induces the binding site for their specific phenolic substrates or their analogs. Based on these findings, a compulsory-ordered kinetic mechanism for the action of these O-methyltransferases is postulated.

Cells, Cultured↗
PubMed1994

Mechanistic studies on human platelet isoprenylated protein methyltransferase: farnesylcysteine analogs block platelet aggregation without inhibiting the methyltransferase.

The kinetic mechanism of the human platelet S-adenosyl-L-methionine (AdoMet)-linked isoprenylated protein methyltransferase was studied and determined to be ordered bibi. AdoMet binds first, and S-adenosyl-L-homocysteine (AdoHcy) departs last. Simple N-acetylated farnesylated cysteine analogs, such as N-acetyl-S-farnesyl-L-cysteine (AFC), are excellent substrates for the enzyme. Although many N-acetylated farnesylated cysteine analogs are excellent substrates for the enzyme, analogs with bulky moieties adjacent to the farnesylcysteine are neither substrates nor inhibitors of the enzyme. Two molecules of this class, N-benzoyl-S-farnesyl-L-cysteine (BzFC) and N-pivaloyl-S-farnesyl-L-cysteine (PFC) are useful in sorting out the putative physiological role of the methyltransferase in mediating human platelet aggregation because their pharmacological activities are unlinked to methyltransferase inhibition. When studied as inhibitors of platelet aggregation, the analogs are as active, or more active, than bona fide methyltransferase inhibitors of similar structure. Therefore, although it is possible that methyltransferase inhibitors, such as AFC, inhibit the enzyme when applied to cells, the observed pharmacological effects appear to be unrelated to this blockade. The new FC analogs described here have revealed a new signal transduction target which will be of some interest to explore.

Animals↗
PubMed2003

(R,S)-Reticuline 7-O-methyltransferase and (R,S)-norcoclaurine 6-O-methyltransferase of Papaver somniferum - cDNA cloning and characterization of methyl transfer enzymes of alkaloid biosynthesis in opium poppy.

S-Adenosyl-L-methionine:(R,S)-reticuline 7-O-methyltransferase converts reticuline to laudanine in tetrahydrobenzylisoquinoline biosynthesis in the opium poppy Papaver somniferum. This enzyme activity has not yet been detected in plants. A proteomic analysis of P. somniferum latex identified a gel spot that contained a protein(s) whose partial amino acid sequences were homologous to those of plant O-methyltransferases. cDNA was amplified from P. somniferum RNA by reverse transcription PCR using primers based on these internal amino acid sequences. Recombinant protein was then expressed in Spodoptera frugiperda Sf9 cells in a baculovirus expression vector. Steady-state kinetic measurements with one heterologously expressed enzyme and mass spectrometric analysis of the enzymatic products suggested that this unusual enzyme is capable of carrying through sequential O-methylations on the isoquinoline and on the benzyl moiety of several substrates. The tetrahydrobenzylisoquinolines (R)-reticuline (4.2 sec(-1) mm(-1)), (S)-reticuline (4.5 sec(-1) mm(-1)), (R)-protosinomenine (1.7 sec(-1) mm(-1)), and (R,S)-isoorientaline (1.4 sec(-1) mm(-1)) as well as guaiacol (5.9 sec(-1) mm(-1)) and isovanillic acid (1.2 sec(-1) mm(-1)) are O-methylated by the enzyme with the ratio kcat/K m shown in parentheses. A P. somniferum cDNA encoding (R,S)-norcoclaurine 6-O-methyltransferase was similarly isolated and characterized. This enzyme was less permissive, methylating only (R,S)-norcoclaurine (7.4 sec(-1) mm(-1)), (R)-norprotosinomenine (4.1 sec(-1) mm(-1)), (S)-norprotosinomenine (4.0 sec(-1) mm(-1)) and (R,S)-isoorientaline (1.0 sec(-1) mm(-1)). A phylogenetic comparison of the amino acid sequences of these O-methyltransferases to those from 28 other plant species suggests that these enzymes group more closely to isoquinoline biosynthetic O-methyltransferases from Coptis japonica than to those from Thalictrum tuberosum that can O-methylate both alkaloid and phenylpropanoid substrates.

Alkaloids↗