Adenosine dialdehyde and neplanocin A: Potent inhibitors of S-adenosylhomocysteine hydrolase in neuroblastoma N2a cells

S-Adenosylhomocysteine hydrolase (AdoHcy hydrolase, E.C. 3.3.1.1) catalyzes the metabolism of S-adenosylhomocysteine (AdoHcy) to adenosine (Ado) and homocysteine (Hcy) in mouse neuroblastoma N2a cells. AdoHcy hydrolase in N2a cells can be inhibited completely by adenosine dialdehyde (Ado dialdehyde) or neplanocin A. The inhibitory effects of Ado dialdehyde (2.5 μM) and neplanocin A (1 μM) on cellular AdoHcy hydrolase were time-dependent, with total enzyme inhibition occurring after 30 min and 15 min of incubation, respectively. The inhibition of AdoHcy hydrolase produced by Ado dialdehyde and neplanocin A persisted for up to 72 h of incubation, and was paralleled by a time-dependent increase in endogenous AdoHcy levels reaching a maximum 4-fold elevation after 8 h of incubation with Ado dialdehyde and an 11-fold increase in the neplanocin A-treated cells. This increase in AdoHcy levels produced a subsequent inhibition of S-adenosylmethionine (AdoMet)-dependent cellular methylations (e.g. protein carboxylmethylation (PCM), lipid methylation). In addition, neplanocin A was metabolically converted to the corresponding AdoMet analog, S-neplanocylmethionine (NepMet), in neuroblastoma N2a cells. NepMet reached maximum levels after 8 h of incubation of the cells with neplanocin A.     

Decreased Transmethylation of Biogenic Amines After In Vivo Elevation of Brain S-Adenosyl-l-Homocysteine

Abstract: The ability of S -adenosyl- l -homocysteine (AdoHcy) to inhibit biologic transmethylation reactions in vitro has led us to explore the possibility of pharmacologically manipulating AdoHcy levels in vivo and examining the consequences of these alterations on the transmethylation of some biogenic amines. Swiss-Webster mice were injected intraperitoneally with different doses of adenosine (Ado) and d,l -homocysteine thiolactone (Hcy) and were killed at various times thereafter. S -Adenosyl- l -methionine (AdoMet) and AdoHcy concentrations were determined by using a modified isotope dilution-ion exchange chromatography-high pressure liquid chromatography technique sensitive to less than 10 pmol. Increasing doses of Ado + Hcy (50-1000 mg/kg of each) produced a dose-related increase in blood, liver, and brain AdoHcy levels. At a dose level of 200 mg/kg Ado + Hcy, AdoHcy levels were markedly elevated, with minimal concomitant perturbations of AdoMet. This elevation was maximal 40 min after giving Ado + Hcy, returning to control values within 6 h. Ado + Hcy treatment resulted in decreased activities of catechol- O -methyltransferase, histamine- N -methyltransferase, and AdoHcy hydrolase in vitro. The cerebral catabolism of intraventricularly administered [³H]histamine (HA) was decreased in a dose-related manner by Ado + Hcy treatment as evidenced by higher amounts of nonutilized [³H]HA in brain, concurrent decreases in [³H]methylhistamine formation, and decreases in the transmethylation conversion index. Steady state levels of HA also showed dose-related increases after Ado + Hcy treatment. It is concluded that injections of Ado + Hcy can markedly elevate AdoHcy levels in vivo , which can, in turn, decrease the rate of transmethylation reactions.     

Adenosine and deoxyadenosine induces apoptosis in oestrogen receptor-positive and -negative human breast cancer cells via the intrinsic pathway

In this study we have examined the cytotoxic effects of different concentrations of adenosine (Ado) and deoxyadenosine (dAdo) on human breast cancer cell lines. Ado and dAdo alone had little effect on cell cytotoxicity. However, in the presence of adenosine deaminase (ADA) inhibitor, EHNA, adenosine and deoxyadenosine led to significant growth inhibition of cells of the lines tested. Ado/EHNA and dAdo/EHNA-induced cell death was significantly inhibited by NBTI, an inhibitor of nucleoside transport, and 5'-amino-5'-deoxyadenosine, an inhibitor of adenosine kinase, but the effects were not affected by 8-phenyltheophylline, a broad inhibitor of adenosine receptors. The Ado/EHNA combination brought about morphological changes consistent with apoptosis. Caspase-9 activation was observed in MCF-7 and MDA-MB468 human breast cancer cell lines on treatment with Ado/EHNA or dAdo/EHNA, but, as expected, caspase-3 activation was only observed in MDA-MB468 cells. The results of the study, thus, suggest that extracellular adenosine and deoxyadenosine induce apoptosis in both oestrogen receptor-positive (MCF-7) and also oestrogen receptor-negative (MDA-MB468) human breast cancer cells by its uptake into the cells and conversion to AMP (dAMP) followed by activation of nucleoside kinase, and finally by the activation of the mitochondrial/intrinsic apoptotic pathway.     

Increase in S-adenosylhomocysteine concentration in interferon-treated HeLa cells and inhibition of methylation of vesicular stomatitis virus mRNA

A fraction of the viral mRNA synthesized in interferon-treated HeLa cells infected with vesicular stomatitis virus (VSV) lacks the 7-methyl group in the 5'-terminal guanosine of the cap; this mRNA is not associated with polyribosomes and does not bind to ribosomes in an assay for initiation of protein synthesis (de Ferra, F., and Baglioni, C. (1981) Virology 112, 426-435). To establish whether this defect in methylation is due to changes in the level of the methyl donor S-adenosylmethionine (AdoMet) and of its competitive inhibitor S-adenosylhomocysteine (AdoHcy), we measured the concentration of these compounds in HeLa cells treated with interferon. An increase in both AdoMet and AdoHcy was detected 3 to 6 h after addition of interferon. The level of these compounds increased gradually and in proportion to the interferon concentration used. With 125 reference units/ml of beta interferon, for example, the AdoHcy concentration increased more than 3-fold and that of AdoMet about 1.5-fold with a consequent change in the AdoHcy/AdoMet ratio. An increased AdoHcy/AdoMet ratio was also found in HeLa cells treated with pure alpha 2 interferon produced in Escherichia coli by recombinant DNA techniques. When the methylation of VSV mRNA was measured in assays carried out with permeabilized virions at the AdoHcy and AdoMet concentrations found in interferon-treated cells, a preferential inhibition of the viral (guanine-7-)methyltransferase activity was observed. Such an inhibition may account for the synthesis of VSV mRNA lacking the 7-methyl group of guanosine in the cap.     

Inhibition of IgE-mediated histamine release from rat basophilic leukemia cells and rat mast cells by inhibitors of transmethylation

This study evaluated the effect of inhibitors of transmethylation on histamine release from rat mast cells and rat basophilic leukemia cells. IgE-mediated histamine release from rat basophilic leukemia cells (RBL-2H3 cells) was inhibited by 3-deazaadenosine (DZA) in the presence of L-homocysteine thiolactone (Hcy) or the combination of adenosine, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), and Hcy in a dose-dependent fashion. There were no significant changes in the cellular cAMP levels by these inhibitors. Histamine release induced by anti-IgE or dextran from normal rat mast cells was also blocked by DZA plus Hcy in a dose-dependent manner. DZA at 10(-3) M in the presence of 10(-4) M Hcy or the combination of 10(-3) M adenosine, 10(-4) M EHNA, and 10(-3) M Hcy inhibited lipid (perhaps phospholipid) methylation into RBL-2H3 cells without affecting choline incorporation. In the presence of 10(-3) M DZA plus 10(-4) M Hcy there was a 170-fold increase in [35S]AdoHcy with the concomitant appearance of 3-deaza-AdoHcy when the cells were incubated with [35S]methionine, thus indicating that these drugs inhibited methylation reaction(s) through the intracellular accumulation of AdoHcy and 3-deaza-AdoHcy. In contrast, histamine release from rat mast cells induced by the calcium ionophore A23187, compound 48/80, polymyxin B, or ATP was not inhibited by these compounds. These results suggest that IgE- or dextran-mediated histamine release involves methylation reactions(s), whereas the other secretagogues bypass this early step.     

Protein Arginine Methylation Is More Prone to Inhibition by S-Adenosylhomocysteine than DNA Methylation in Vascular Endothelial Cells

Methyltransferases use S-adenosylmethionine (AdoMet) as methyl group donor, forming S-adenosylhomocysteine (AdoHcy) and methylated substrates, including DNA and proteins. AdoHcy inhibits most methyltransferases. Accumulation of intracellular AdoHcy secondary to Hcy elevation elicits global DNA hypomethylation. We aimed at determining the extent at which protein arginine methylation status is affected by accumulation of intracellular AdoHcy. AdoHcy accumulation in human umbilical vein endothelial cells was induced by inhibition of AdoHcy hydrolase by adenosine-2,3-dialdehyde (AdOx). As a measure of protein arginine methylation status, the levels of monomethylarginine (MMA) and asymmetric and symmetric dimethylated arginine residues (ADMA and SDMA, respectively) in cell protein hydrolysates were measured by HPLC. A 10% decrease was observed at a 2.5-fold increase of intracellular AdoHcy. Western blotting revealed that the translational levels of the main enzymes catalyzing protein arginine methylation, protein arginine methyl transferases (PRMTs) 1 and 5, were not affected by AdoHcy accumulation. Global DNA methylation status was evaluated by measuring 5-methylcytosine and total cytosine concentrations in DNA hydrolysates by LC-MS/MS. DNA methylation decreased by 10% only when intracellular AdoHcy concentration accumulated to 6-fold of its basal value. In conclusion, our results indicate that protein arginine methylation is more sensitive to AdoHcy accumulation than DNA methylation, pinpointing a possible new player in methylation-related pathology.     

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.     

The methyl donor S-Adenosylmethionine inhibits active demethylation of DNA: a candidate novel mechanism for the pharmacological effects of S-Adenosylmethionine

S-Adenosylmethionine (AdoMet) is the methyl donor of numerous methylation reactions. The current model is that an increased concentration of AdoMet stimulates DNA methyltransferase reactions, triggering hypermethylation and protecting the genome against global hypomethylation, a hallmark of cancer. Using an assay of active demethylation in HEK 293 cells, we show that AdoMet inhibits active demethylation and expression of an ectopically methylated CMV-GFP (green fluorescent protein) plasmid in a dose-dependent manner. The inhibition of GFP expression is specific to methylated GFP; AdoMet does not inhibit an identical but unmethylated CMV-GFP plasmid. S-Adenosylhomocysteine (AdoHcy), the product of methyltransferase reactions utilizing AdoMet does not inhibit demethylation or expression of CMV-GFP. In vitro, AdoMet but not AdoHcy inhibits methylated DNA-binding protein 2/DNA demethylase as well as endogenous demethylase activity extracted from HEK 293, suggesting that AdoMet directly inhibits demethylase activity, and that the methyl residue on AdoMet is required for its interaction with demethylase. Taken together, our data support an alternative mechanism of action for AdoMet as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA.     

Inhibition of EcoRI DNA methylase with cofactor analogs

Four analogs of the natural cofactor S-adenosylmethionine (AdoMet) were tested for their ability to bind and inhibit the prokaryotic enzyme, EcoRI adenine DNA methylase. The EcoRI methylase transfers the methyl group from AdoMet to the second adenine in the double-stranded DNA sequence 5'GAATTC3'. Dissociation constants (KD) of the binary methylase-analog complexes obtained in the absence of DNA with S-adenosylhomocysteine (AdoHcy), sinefungin, N-methyl-AdoMet, and N-ethylAdoMet are 225, 43, greater than 1000, and greater than 1000 microM, respectively. In the presence of a DNA substrate, all four analogs show simple competitive inhibition with respect to AdoMet. The product of the enzymic reaction, AdoHcy, is a poor inhibitor of the enzyme (KI(AdoHcy) = 9 microM; KM(AdoMet) = 0.60 microM). Two synthetic analogs, N-methyl-AdoMet and N-ethyl-AdoMet, were also shown to be poor inhibitors with KI values of 50 and greater than 1000 microM, respectively. In contrast, the naturally occurring analog sinefungin was shown to be a highly potent inhibitor (KI = 10 nM). Gel retardation assays confirm that the methylase-DNA-sinefungin complex is sequence-specific. The ternary complex is the first sequence-specific complex detected for any DNA methylase. Potential applications to structural studies of methylase-DNA interactions are discussed.     

Computational characterization of substrate binding and catalysis in S-adenosylhomocysteine hydrolase.

S-Adenosylhomocysteine (AdoHcy) hydrolase catalyzes the reversible hydrolysis of AdoHcy to adenosine (Ado) and homocysteine (Hcy), playing an essential role in modulating the cellular Hcy levels and regulating activities of a host of methyltransferases in eukaryotic cells. This enzyme exists in an open conformation (active site unoccupied) and a closed conformation (active site occupied with substrate or inhibitor) [Turner, M. A., Yang, X., Yin, D., Kuczera, K., Borchardt, R. T., and Howell, P. L. (2000) Cell Biochem. Biophys. 33, 101-125]. To investigate the binding of natural substrates during catalysis, the computational docking program AutoDock (with confirming calculations using CHARMM) was used to predict the binding modes of various substrates or inhibitors with the closed and open forms of AdoHcy hydrolase. The results have revealed that the interaction between a substrate and the open form of the enzyme is nonspecific, whereas the binding of the substrate in the closed form is highly specific with the adenine moiety of a substrate as the main recognition factor. Residues Thr57, Glu59, Glu156, Gln181, Lys186, Asp190, Met351, and His35 are involved in substrate binding, which is consistent with the crystal structure. His55 in the docked model appears to participate in the elimination of water from Ado through the interaction with the 5'-OH group of Ado. In the same reaction, Asp131 removes a proton from the 4' position of the substrate after the oxidation-reduction reaction in the enzyme. To identify the residues that bind the Hcy moiety, AdoHcy was docked to the closed form of AdoHcy hydrolase. The Hcy tail is predicted to interact with His55, Cys79, Asn80, Asp131, Asp134, and Leu344 in a strained conformation, which may lower the reaction barrier and enhance the catalysis rate.     

Two alternative conformations of S-adenosyl-L-homocysteine bound to Escherichia coli DNA adenine methyltransferase and the implication of conformational changes in regulating the catalytic cycle

The crystal structure of the Escherichia coli DNA adenine methyltransferase (EcoDam) in a binary complex with the cofactor product S-adenosyl-L-homocysteine (AdoHcy) unexpectedly showed the bound AdoHcy in two alternative conformations, extended or folded. The extended conformation represents the catalytically competent conformation, identical to that of EcoDam-DNA-AdoHcy ternary complex. The folded conformation prevents catalysis, because the homocysteine moiety occupies the target Ade binding pocket. The largest difference between the binary and ternary structures is in the conformation of the N-terminal hexapeptide ((9)KWAGGK(14)). Cofactor binding leads to a strong change in the fluorescence of Trp(10), whose indole ring approaches the cofactor by 3.3A(.) Stopped-flow kinetics and AdoMet cross-linking studies indicate that the cofactor prefers binding to the enzyme after preincubation with DNA. In the presence of DNA, AdoMet binding is approximately 2-fold stronger than AdoHcy binding. In the binary complex the side chain of Lys(14) is disordered, whereas Lys(14) stabilizes the active site in the ternary complex. Fluorescence stopped-flow experiments indicate that Lys(14) is important for EcoDam binding of the extrahelical target base into the active site pocket. This suggests that the hexapeptide couples specific DNA binding (Lys(9)), AdoMet binding (Trp(10)), and insertion of the flipped target base into the active site pocket (Lys(14)).     

Enhancement of macrophage Fc-dependent phagocytosis by resident thymocytes: effect of a unique heat-stable lymphokine

Lymphocytes, activated by lectins or specific antigens, have been shown to enhance macrophage phagocytosis through the elaboration of a heat-labile soluble factor(s). Recent evidence from our laboratory revealed that resident (nonactivated) murine thymocytes and splenic lymphocytes increase peritoneal macrophage glucose metabolism through the elaboration of a heat-stable soluble factor(s). Therefore, we investigated the effect of resident lymphocyte subpopulations on macrophage Fc-dependent phagocytosis. Thioglycollate-elicited and resident peritoneal macrophages from BALB/c mice were cultured in serum-free media with syngeneic resident thymocytes or splenic T lymphocytes. Macrophage Fc-dependent phagocytosis was assayed by measuring the ingestion of 51CrSHEA. After 4 days in vitro, resident thymocytes produced a mean 160 (+/- 31) and 136% (+/- 22) increase in Fc-dependent phagocytosis by thioglycollate-elicited (thio-macrophages) and resident peritoneal macrophages, respectively. Splenic T lymphocytes increased thio-macrophage phagocytosis by 112% (+/- 41) under similar conditions. Macrophage Fc-dependent phagocytosis was increased after 24 hr of co-culture by supernatant derived from resident thymocytes and could be further enhanced by supernatant from Con A-activated thymocytes. Supernatant from guinea pig embryo fibroblasts did not increase macrophage phagocytosis. The soluble factor(s) was produced by resident thymocytes after 24 hr of preculture. This factor was active despite heating at 100 degrees C for 30 min whereas the effect of Con A-activated thymocyte supernatant was heat-labile. The stimulatory effect of resident thymocyte supernatant was not observed when the macrophages and supernatant were cultured in 2% FCS. In contrast to the factor(s) produced by resident thymocytes, the factor(s) in FCS that increased phagocytosis was heat-labile. These data suggest thymocytes and splenic T lymphocytes promote macrophage Fc-dependent phagocytosis in the absence of antigenic or lectin stimulation. This previously unrecognized effect of resident thymocytes is due to a unique heat-stable soluble factor(s) that is concealed in the presence of serum.     

Inhibitory effect of Epimedium extract on S-adenosyl-L-homocysteine hydrolase and biomethylation

In the present paper, the inhibitory effect of Epimedium extract on the activity of S-adenosyl-L-homocysteine (AdoHcy) Hydrolase was studied. The results showed that Epimedium extract inhibited the activity of recombinant human AdoHcy hydrolase in a dose-dependent manner. This inhibitory effect was also observed in hepatic cell line 7701 and hepatoma HepG2, however, the effect in 7701 cells was more potent than in HepG2 cells. The extract could significantly reduce AdoMet/AdoHcy ratio in 7701 cells in a dose-dependent manner, suggesting reduced biomethylation level in 7701 cells. In contrast, it resulted in elevated AdoMet/AdoHcy ratio in the HepG2 cells. The result of MALDI-MS assay indicated that epimedin A and ikarisoside F from the extract could bind to AdoHcy hydrolase. The present data suggested that Epimedium extract could inhibit the activity of AdoHcy hydrolase, thus regulating the cellular biomethylation as well as reducing cellular Hcy level. These results will provide new clues to the mechanisms of Epimedium in curing of cardiovascular disease and regulating tumor cell growth.     

Bacteriophage T4 Dam DNA-[N6-adenine]methyltransferase. Kinetic evidence for a catalytically essential conformational change in the ternary complex.

We carried out a steady state kinetic analysis of the bacteriophage T4 DNA-[N6-adenine]methyltransferase (T4 Dam) mediated methyl group transfer reaction from S-adenosyl-l-methionine (AdoMet) to Ade in the palindromic recognition sequence, GATC, of a 20-mer oligonucleotide duplex. Product inhibition patterns were consistent with a steady state-ordered bi-bi mechanism in which the order of substrate binding and product (methylated DNA, DNA(Me) and S-adenosyl-l-homocysteine, AdoHcy) release was AdoMet downward arrow DNA downward arrow DNA(Me) upward arrow AdoHcy upward arrow. A strong reduction in the rate of methylation was observed at high concentrations of the substrate 20-mer DNA duplex. In contrast, increasing substrate AdoMet concentration led to stimulation in the reaction rate with no evidence of saturation. We propose the following model. Free T4 Dam (initially in conformational form E) randomly interacts with substrates AdoMet and DNA to form a ternary T4 Dam-AdoMet-DNA complex in which T4 Dam has isomerized to conformational state F, which is specifically adapted for catalysis. After the chemical step of methyl group transfer from AdoMet to DNA, product DNA(Me) dissociates relatively rapidly (k(off) = 1.7 x s(-1)) from the complex. In contrast, dissociation of product AdoHcy proceeds relatively slowly (k(off) = 0.018 x s(-1)), indicating that its release is the rate-limiting step, consistent with kcat = 0.015 x s(-1). After AdoHcy release, the enzyme remains in the F conformational form and is able to preferentially bind AdoMet (unlike form E, which randomly binds AdoMet and DNA), and the AdoMet-F binary complex then binds DNA to start another methylation cycle. We also propose an alternative pathway in which the release of AdoHcy is coordinated with the binding of AdoMet in a single concerted event, while T4 Dam remains in the isomerized form F. The resulting AdoMet-F binary complex then binds DNA, and another methylation reaction ensues. This route is preferred at high AdoMet concentrations.     

Bacteriophage T4Dam DNA-(adenine-N(6))-methyltransferase. Comparison of pre-steady state and single turnover methylation of 40-mer duplexes containing two (un)modified target sites

We analyzed pre-steady state and single turnover kinetics of bacteriophage T4Dam DNA-(adenine-N(6))-methyltransferase-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to 40-mer duplexes containing native recognition sites (5'-GATC/5'-GATC) or some modified variant(s). The results extend a model from studies with single-site 20-mer duplexes. Under pre-steady state conditions, monomeric T4Dam methyltransferase-AdoMet complexes were capable of rapid methylation of adenine residues in 40-mer duplexes containing two sites. During processive movement of T4Dam to the next site, the rate-limiting step was the exchange of the product S-adenosyl-l-homocysteine (AdoHcy) for AdoMet without T4Dam dissociating from the duplex. Consequently, instead of a single exponential rate dependence, complex methylation curves were obtained with at least two pre-steady state steps. With 40-mer duplexes containing a single target site, the kinetics were simpler, fitting a single exponential followed by a linear steady state phase. Single turnover methylation of 40-mer duplexes also proceeded in two stages. First, two dimeric T4Dam-AdoMet molecules bound, and each catalyzed a two-step methylation. Instead of processive movement of T4Dam, a conformational adaptation occurred. We propose that following methyl transfer to one strand, dimeric (T4Dam-AdoMet)-(T4Dam-AdoHcy) was capable of rapidly reorienting itself and catalyzing methyl transfer to the target adenine on the complementary, unmethylated strand. This second stage methyl transfer occurred at a rate about 25-fold slower than in the first step; it was rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand. Under single turnover conditions, there was complete methylation of all target adenine residues with each of the two-site 40-mer duplexes.     

S-Adenosyl-Homocysteine Is a Weakly Bound Inhibitor for a Flaviviral Methyltransferase

The methyltransferase enzyme (MTase), which catalyzes the transfer of a methyl group from S-adenosyl-methionine (AdoMet) to viral RNA, and generates S-adenosyl-homocysteine (AdoHcy) as a by-product, is essential for the life cycle of many significant human pathogen flaviviruses. Here we investigated inhibition of the flavivirus MTase by several AdoHcy-derivatives. Unexpectedly we found that AdoHcy itself barely inhibits the flavivirus MTase activities, even at high concentrations. AdoHcy was also shown to not inhibit virus growth in cell-culture. Binding studies confirmed that AdoHcy has a much lower binding affinity for the MTase than either the AdoMet co-factor, or the natural AdoMet analog inhibitor sinefungin (SIN). While AdoMet is a positively charged molecule, SIN is similar to AdoHcy in being uncharged, and only has an additional amine group that can make extra electrostatic contacts with the MTase. Molecular Mechanics Poisson-Boltzmann Sovation Area analysis on AdoHcy and SIN binding to the MTase suggests that the stronger binding of SIN may not be directly due to interactions of this amine group, but due to distributed differences in SIN binding resulting from its presence. The results suggest that better MTase inhibitors could be designed by using SIN as a scaffold rather than AdoHcy.     

S-adenosyl-L-homocysteine hydrolase and methylation disorders: Yeast as a model system

S-adenosyl-L-methionine (AdoMet)-dependent methylation is central to the regulation of many biological processes: more than 50 AdoMet-dependent methyltransferases methylate a broad spectrum of cellular compounds including nucleic acids, proteins and lipids. Common to all AdoMet-dependent methyltransferase reactions is the release of the strong product inhibitor S-adenosyl-L-homocysteine (AdoHcy), as a by-product of the reaction. S-adenosyl-L-homocysteine hydrolase is the only eukaryotic enzyme capable of reversible AdoHcy hydrolysis to adenosine and homocysteine and, thus, relief from AdoHcy inhibition. Impaired S-adenosyl-L-homocysteine hydrolase activity in humans results in AdoHcy accumulation and severe pathological consequences. Hyperhomocysteinemia, which is characterized by elevated levels of homocysteine in blood, also exhibits a similar phenotype of AdoHcy accumulation due to the reversal of the direction of the S-adenosyl-L-homocysteine hydrolase reaction. Inhibition of S-adenosyl-L-homocysteine hydrolase is also linked to antiviral effects. In this review the advantages of yeast as an experimental system to understand pathologies associated with AdoHcy accumulation will be discussed.