Methionine biosynthesis in isolated Pisum sativum mitochondria

Homocysteine-dependent transmethylases utilizing 5-methyltetrahydropteroylglutamic acid and S-adenosylmethionine as methyl donors have been examined using ammonium sulphate fractions prepared from isolated mitochondria of pea cotyledons. Substantial levels of a 5-rnethyltetrahydropteroylglutamate transmethylase were detected, the catalytic properties of this enzyme being found similar to those of a previously reported enzyme present in cotyledon extracts. The mitochondrial 5-CH₃-H₄PteGlu transmethylase had an apparent K_m of 25 μM for the methyl donor, was saturated with homocysteine at 1 mM and was inhibited 50% by l-methionine at 2.5 mM. At similar concentrations of methyl donor the mitochondrial S-adenosylmethionine methyltransferase was not saturated. Mitochondrial preparations were found capable of synthesizing substantial amounts of S-adenosylmethionine but lacked ability to form S-methylmethionine. Significant levels of β-cystathionase, cystathionine-γ-synthase, l-homoserine transacetylase and l-homoserine transsuccinylase were detected in the isolated mitochondria. The activity of the enzymes of homocysteine biosynthesis was not affected by l-methionine in vitro. It is concluded that pea mitochondria have ability to catalyze the synthesis of methionine de novo.     

Purification and properties of caffeic acid O-methyltransferase from alfalfa root nodules

An O-methyltransferase which catalyses the methylation of caffeic acid to ferulic acid using S-adenosyl-l-methionine as methyl donor has been isolated and purified ca 70-fold from root nodules of alfalfa. The enzyme also catalysed the methylation of 5-hydroxyferulic acid. Chromatography on 1,6-diaminohexane agarose (AH-Sepharose-4B) linked with S-adenosyl-l-homocysteine (SAH) gave 35% recovery of enzyme activity. The K_m values for caffeic acid and S-adenosyl-l-methionine were 58 and 4.1 μM, respectively. S-Adenosyl-l-homocysteine was a potent competitive inhibitor of S-adenosyl-l-methionine with a K_i of 0.44 μM. The MW of the enzyme was ca 103 000 determined by gel filtration chromatography.     

Enzymic synthesis of lignin precursors. Purification of properties of the S-adenosyl-l-methionine: caffeic acid 3-O- methyltransferase from soybean cell suspension cultures.

A 3-O-methyltransferase which catalyzes the methylation of caffeic acid to ferulic acid using S-adenosyl-l-methionine as methyl donor has been isolated and purified about 60-fold from cell suspension cultures of soybean (Glycine max L., var. Mandarin). The enzyme utilized, in addition to caffeic acid (K_m = 133 μM), 5-hydroxyferulic acid (K_m = 55 μM), 3,4,5-trihydroxy-cinnamic acid (K_m = 100 μM), and protocatechualdehyde (K_m = 50 μM) as substrates. Methylation proceeded only in the meta position. The enzyme was unable to catalyze the methylation of ferulic acid, of ortho-, meta-, and para-coumaric acids, and of the flavonoid compounds quercetin and luteolin. The methylation of caffeic acid and 5-hydroxyferulic acid showed a pH optimum at 6.5–7.0. No stimulation of the reaction velocity was observed when Mg²⁺ ions were added. EDTA did not inhibit the reaction. The K_m for S-adencsyl-l-methionine was 15 μm. S-Adenosyl-l-homocysteine was a potent competitive inhibitor of S-adenosyl-l-methionine (K_i = 6.9 μM).     

Using S-adenosyl-l-homocysteine capture compounds to characterize S-adenosyl-l-methionine and S-adenosyl-l-homocysteine binding proteins

S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH–CC with biotin used in conjunction with streptavidin–horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the K_d values for COMT, SAHH, and PRDM2 (24.1 ± 2.2 μM, 6.0 ± 2.9 μM, and 10.06 ± 2.87 μM, respectively) and found them to be close to previously established K_d values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs.     

Electric field-induced changes in agonist-stimulated calcium fluxes of human HL-60 leukemia cells

The mechanism of biological effects of extremely-low-frequency electric and magnetic fields may involve induced changes of Ca²⁺ transport through plasma membrane ion channels. In this study we investigated the effects of externally applied, low-intensity 60 Hz electric (E) fields (0.5 V/m, current density 0.8 A/m²⁺) on the agonist-induced Ca²⁺ fluxes of HL-60 leukemia cells. The suspensions of HL-60 cells received E-field or sham exposure for 60 min and were simultaneously stimulated either by 1 μM ATP or by 100 μM histamine or were not stimulated at all. After E-field or sham exposure, the responses of the intracellular calcium levels of the cells to different concentrations of ATP (0.2–100 μM) were assessed. Compared with control cells, exposure of ATP-activated cells to an E-field resulted in a 20–30% decrease in the magnitude of [Ca²⁺]_i elevation induced by a low concentration of ATP (<1 μM). In contrast, exposure of histamine-activated HL-60 cells resulted in a 20–40% increase of ATP-induced elevation of [Ca²⁺]_i. E-field exposure had no effect on non-activated cells. Kinetic analysis of concentration-response plots also showed that compared with control cells, exposure to the E-field resulted in increases of the Michaelis constant, K_m, value in ATP-treated cells and of the maximal [Ca²⁺]_i peak rise in histamine-treated HL-60 cells. The observed effects were reversible, indicating the absence of permanent structural damages induced by acute 60 min exposure to electric fields. These results demonstrate that low-intensity electric fields can alter calcium distribution in cells, most probably due to the effect on receptor-operated Ca²⁺ and/or ion channels. Bioelectromagnetics 19:366–376, 1998. © 1998 Wiley-Liss, Inc.     

1-Aminocyclopropanecarboxylate synthase, a key enzyme in ethylene biosynthesis

1-Aminocyclopropanecarboxylate (ACC) synthase, which catalyzes the conversion of S-adenosylmethionine (SAM) to ACC and methylthioadenosine, was demonstrated in tomato extract. Methylthioadenosine was then rapidly hydrolyzed to methylthioribose by a nucleosidase present in the extract. ACC synthase had an optimum pH of 8.5, and a K_m of 20 μm with respect to SAM. S-Adenosylethionine also served as a substrate for ACC synthase, but at a lower efficiency than that of SAM. Since S-adenosylethionine had a higher affinity for the enzyme than SAM, it inhibited the reaction of SAM when both were present. S-Adenosylhomocysteine was, however, an inactive substrate. The enzyme was activated by pyridoxal phosphate at a concentration of 0.1 μm or higher, and competitively inhibited by aminoethoxyvinylglycine and aminooxyacetic acid, which are known to inhibit pyridoxal phosphate-mediated enzymic reactions. These results support the view that ACC synthase is a pyridoxal enzyme. The biochemical role of pyridoxal phosphate is catalyzing the formation of ACC by α,γ-elimination of SAM is discussed.     

Effect of adenosylhomocysteine and other analog thioethers on a prokaryotic tRNA (guanine-7)-methyltransferase

S-Adenosylhomocysteine (SAH), a potent inhibitor of methyltransferases, and several thioethers structurally related to SAH, have been tested as potential inhibitors of tRNA (guanine-7)-methyltransferase from Salmonella typhimurium. The tested compounds are l-, d-, dl-S-adenosylhomocysteine, S-adenosylcysteine, methylthioadenosine, butylthioadenosine, thioethanoladenosine, isobutylthioadenosine, S-inosylhomocysteine, and methylthioinosine. Among them the highest inhibitory activity has been shown by SAH (K_i = 8 μM), whereas butylthioadenosine, isobutylthioadenosine, and thioethanoladenosine are almost inactive as inhibitors. The other compounds inhibit the enzyme with K_i values ranging between 400 and 800 μm. From these data it is possible to evaluate the importance of the -NH₂ and -COOH groups of the substrate in the binding to the enzyme molecule, as well as other features such as the chirality at the α-carbon atom and the length of the hydrocarbon chain connecting the -NH₂ and -COOH groups to the aromatic ring of adenosine. The aminic group of the adenosine is also critical, because S-inosylhornocysteine and methylthioinosine are poorer inhibitors in comparison with SAH and methylthioadenosine.     

Purification and partial characterization of the glutathione S-transferase of rat erythrocytes

The single glutathione S-transferase (EC 2.5.1.18) present in rat erythrocytes was purified to apparent homogeneity by affinity chromatography on glutathione-Sepharose and hydroxyapatite chromatography. Approx. 1.86 mg enzyme is found in 100 ml packed erythrocytes and accounts for about 0.01% of total soluble protein. The native enzyme (M_r 48 000) displays a pI of 5.9 and appears to possess a homodimeric structure with a subunit of M_r 23 500. Enzyme activities with ethacrynic acid and cumene hydroperoxide were 24 and 3%, respectively, of that with 1-chloro-2,4-dinitrobenzene. The K_m values for 1-chloro-2,4-dinitrobenzene and glutathione were 1.0 and 0.142 mM, respectively. The concentrations of certain compounds required to produce 50% inhibition (I₅₀) were as follows: 12 μM bromosulphophthalein, 34 μM S-hexylglutathione, 339 μM oxidized glutathione and 1.5 mM cholate. Bromosulphophthalein was a noncompetitive inhibitor with respect to 1-chloro-2,4-dinitrobenzene (K_i = 8 μM) and glutathione (K_is = 4 μM; K_ii = 11.5 μM) while S-hexylglutathione was competitive with glutathione (K_i = 5 μM).     

Membrane-bound O-methyltransferase of douglas-fir callus

A lignin-specific O-methyltransferase (OMT) was localized in the cell wall fraction of Douglas-fir needle callus homogenates. The OMT was released from wall-associated membrane by digitonin and partially purified by salt fractionation. Further purification proved to be unfeasible, due to the high tannin content of the callus. The K_m values of the partially purified OMT for caffeic acid and S-adenosylmethionine (SAM) were 250 and 8.0.μM, respectively. Substrate inhibition as well as inhibition by S-adenosylhomocysteine (SAH) was observed. Coupled with low levels of caffeic acid found in the callus, 65,μM at maximum with a mean of 11.5μM throughout a subculture period, the properties of this OMT should account in large part for the high tannin and low lignin content characteristic of this cultured tissue.     

Investigation of the subcellular distribution of cyclic-AMP phosphodiesterase in rat hepatocytes,using a rapid immunological procedure for the isolation of plasma membrane

The distribution of cyclic-AMP phosphodiesterase was investigated in subcellular fractions prepared from homogenates of rat liver or isolated hepatocytes. When measured at 1 mM or 1 μM substrate concentration, approx. 35% or 50%, respectively, of enzyme activity was particulate. The soluble activity appeared to be predominantly a ‘high K_m’ form, whereas the particulate activity had both ‘high K_m’ and ‘low K_m’ components. The recovery of cyclic-AMP phosphodiesterase was measured using 1 μM substrate concentration, in plasma membrane-containing fractions prepared either by centrifugation or by the use of specific immunoadsorbents. The recovery of phosphodiesterase was lower than that of marker enzymes for plasma membrane, and comparable with the recovery of markers for intracellular membranes. It was concluded that regulation of both ‘high K_m’ and ‘low K_m’ phosphodiesterase could potentially make a significant contribution to the control of cyclic AMP concentration, even at μM levels, in the liver. The ‘low K_m’ enzyme, for which activation by hormones has been previously described, appears to be located predominantly in intracellylar membranes in hepatocytes.The immunological procedure for membrane isolation allowed the rapid preparation of plasma membranes in high yield. Liver cells were incubated with rabbit anti-(rat erythrocyte) serum and homogenized. The antibody-coated membrane fragments were then extracted onto an immunoadsorbent consisiting of sheep anti-(rabbit IgG) immunoglobulin covalently bound to aminocellulose. Plasma membrane was obtained in approx. 40% yield within 50 min of homogenizing cells.     

Activity and kinetic properties of basal aryl hydrocarbon hydroxylase during proliferation in the transformable C3H 10T12; CL8 cell line

Basal aryl hydrocarbon hydroxylase (AHH) activity and its kinetic properties were studied as a function of proliferation in C3H mouse embryo 10T$\text{1}\text{2}$ CL8 cells. Activity was low in freshly plated cells, increased during exponential growth, peaked at confluency, and then declined. The apparent K_m-values for benzo[a]pyrene (BP) and NADPH were less in proliferating (approx. 0.37 μM BP, 3.3 nM NADPH) than in confluent cells (0.74–1.39 μM BP, 33.4–53.4 nM NADPH). Cells at different growth states responded differently to benz[a]anthracene (BA) and aminophylline, an inhibitor of cyclic nucleotide phosphodiesterases. When cells were harvested at the mid log phase of growth, 12 h of exposure to aminophylline caused maximum induction, while 24 h of BA treatment were required. In contrast, at early confluence, 12 h of BA treatment gave the greatest levels of activity, while exposure to aminophylline did not induce AHH. In fact, decreases in activity were observed. These differences are indicative of different regulatory mechanisms for BA and aminophylline induction. They also suggest the regulation of basal AHH by cyclic nucleotides changes during growth. The exposure times giving maximum activity were used to determine the kinetic properties of BA-induced activity. As with basal AHH, the K_m-value for BP was less in log phase (0.2–0.4 μM BP) than in confluent cells (0.64–1.05 μM BP). Moreover, the K_m-values for BP and NADPH in control cultures at confluency (0.10–0.14 μM BP, 15.4–23.2 nM NADPH) were less than those for BA-treated cells (0.64 μM BP, 37.9–54.8 nM NADPH) under the same nutritional conditions. The finding that the K_m-value for BP is lower in rapidly dividing cells than in confluent cells may help to explain why proliferating cells are more susceptible to transforming agents.     

A sensitive mass spectrum assay to characterize engineered methionine adenosyltransferases with S-alkyl methionine analogues as substrates

Methionine adenosyltransferases (MATs) catalyze the formation of S-adenosyl-l-methionine (SAM) inside living cells. Recently, S-alkyl analogues of SAM have been documented as cofactor surrogates to label novel targets of methyltransferases. However, these chemically synthesized SAM analogues are not suitable for cell-based studies because of their poor membrane permeability. This issue was recently addressed under a cellular setting through a chemoenzymatic strategy to process membrane-permeable S-alkyl analogues of methionine (SAAMs) into the SAM analogues with engineered MATs. Here we describe a general sensitive activity assay for engineered MATs by converting the reaction products into S-alkylthioadenosines, followed by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) quantification. With this assay, 40 human MAT mutants were evaluated against 7 SAAMs as potential substrates. The structure–activity relationship revealed that, besides better engaged SAAM binding by the MAT mutants (lower K_m value in contrast to native MATs), the gained activity toward the bulky SAAMs stems from their ability to maintain the desired linear S_N2 transition state (reflected by higher k_cat value). Here the I117A mutant of human MATI was identified as the most active variant for biochemical production of SAM analogues from diverse SAAMs.     

Regulation of ornithine decarboxylase activity in Mucor bacilliformis and Mucor rouxii

Ornithine decarboxylase (ODC) from Mucor bacilliformis and Mucor rouxii was studied. Enzymatic activity was maximal at pH 7.2–7.4 and at 30°C. The K_m was 0.17 mM for the M. bacilliformis enzyme. Putrescine was a competitive inhibitor of ODC with a K_i of 2–3 mM. Enzymatic activity was undetectable in sporangiospores but increased rapidly during the first stages of spore swelling, reaching the highest levels during germ tube or bud emergence, and then decreased. Incubation at 30°C inhibited spore germination in M. bacilliformis and prevented development of ODC activity. More ODC activity was present in mycelial than in yeast cells. Morphological transition of yeast cells into hyphae by an anaerobic-aerobic shift induced a rapid and transient increase in ODC activity. Similar results were obtained when the morphogenetic transformation of M. rouxii was induced by CO₂ elimination in an anaerobic environment. Transfer of mycelial cells to anaerobiosis resulted in a rapid decrease in enzyme activity. Changes in ODC activity were accompanied by a change in the pool of polyamines. The possible role of ODC in growth and cell differentiation in Mucor is discussed.     

Adenine nucleotide metabolism of blood platelets. VIII. Transport of adenine into human platelets

Adenine uptake into human blood platelets is a carrier-mediated process with a K_m of 159±21 nM and a V of 100±10 pmoles/min per 10⁹ platelets (in citrated platelet-rich plasma). The Q₁₀ was 2.53±0.22. A pH optimum was found at 7.5. Washing of the platelets increased the K_m to 453±33 nM and V to 397±38 pmoles/min per 10⁹ platelets. The change in shape induced in platelets by ADP was accompanied by an increase in V (2 times) and K_m (1.5 times).Guanine (K_i 50 μM), hypoxanthine (K_i 390 μM), adenine-N′-oxide (K_i 40 μM), adenosine (K_i 100 μM), RA 233 (K_i 75 μM) and papaverine (K_i 15 μM) acted as competitive inhibitors. Adenosine at low concentrations, and prostaglandin E₁ gave inhibition at only high adenine levels. A similar inhibition was obtained with 2-deoxy-d-glucose. Sulfhydryl-group inhibitors, pyrimidines and ouabain had no effect.     

Reversed-phase high-performance liquid chromatography procedure for the simultaneous determination of S-adenosyl-l-methionine and S-adenosyl-l-homocysteine in mouse liver and the effect of methionine on their concentrations

An improved reversed-phase high-performance liquid chromatography (HPLC) procedure with ultraviolet detection is described for the simultaneous determination of S-adenosyl-l-methionine (SAM) and S-adenosyl-l-homocysteine (SAH) in mouse tissue. The method provides rapid resolution of both compounds in a 25-μl perchloric acid extract of the tissue. The limits of detection in 25-μl injection volumes were 22 and 20 pmol for SAM and SAH, respectively. The limits of quantitation in 25-μl injection volumes were 55 and 50 pmol for SAM and SAH, respectively, with recovery consistently >98%. The assay was validated over linear ranges of 55–11 000 pmol for SAM and 50–10 000 pmol for SAH. The intra-day precision and accuracy were ≤6.4% relative standard deviation (RSD) and 99.9–100.0% for SAH and ≤6.7% RSD and 100.0–100.1% for SAM. The inter-day precision and accuracy were ≤5.9% RSD and 99.9–100.6% for SAH and ≤7.0% RSD and 99.5–100.1% for SAM. Compared to earlier procedures, the HPLC method demonstrated significantly better separation, detection limit and linear range for SAM and SAH determination. The assay demonstrated applicability to monitoring in mice the time-course of the effect of methionine on SAM and SAH levels in the liver. Administering methionine to mice increased by 10-fold the liver concentration of SAM and SAH within 2 h, which then rapidly decreased to the control levels by 8 h. This indicated that methionine was promptly converted to SAM and then rapidly catabolized into SAH. Thus, the metabolism of methionine to SAM should be considered in the supplementation of methionine to maintain SAM levels in the body.     

Identification of a 5′-Deoxyadenosine Deaminase in Methanocaldococcus jannaschii and Its Possible Role in Recycling the Radical S-Adenosylmethionine Enzyme Reaction Product 5′-Deoxyadenosine

We characterize here the MJ1541 gene product from Methanocaldococcus jannaschii, an enzyme that was annotated as a 5′-methylthioadenosine/S-adenosylhomocysteine deaminase (EC 3.5.4.31/3.5.4.28). The MJ1541 gene product catalyzes the conversion of 5′-deoxyadenosine to 5′-deoxyinosine as its major product but will also deaminate 5′-methylthioadenosine, S-adenosylhomocysteine, and adenosine to a small extent. On the basis of these findings, we are naming this new enzyme 5′-deoxyadenosine deaminase (DadD). The K_m for 5′-deoxyadenosine was found to be 14.0 ± 1.2 μM with a k_cat/K_m of 9.1 × 10⁹ M⁻¹ s⁻¹. Radical S-adenosylmethionine (SAM) enzymes account for nearly 2% of the M. jannaschii genome, where the major SAM derived products is 5′-deoxyadenosine. Since 5′-dA has been demonstrated to be an inhibitor of radical SAM enzymes; a pathway for removing this product must be present. We propose here that DadD is involved in the recycling of 5′-deoxyadenosine, whereupon the 5′-deoxyribose moiety of 5′-deoxyinosine is further metabolized to deoxyhexoses used for the biosynthesis of aromatic amino acids in methanogens.     

5-methyletrahydrohomofolate: a substrate for cobalamin methyltransferases and an inhibitor of cell growth

5-Methyltetrahydrohomofolate (5-MeH₄-homofolate) is a substrate for the cobalamin (B-12) methyltransferases in Escherichia coli B, rabbit liver, HeLa S₃ cells, and Chinese hamster ovary cells. Each of these B-12 enzymes catalyzes 5-MeH₄-homofolate-homocysteine transmethylation at one-tenth the rate of methionine synthesis from 5-MeH₄-folate. Only one stereoisomer in dl-5-MeH₄-homofolate is active enzymically. Reduced higher 5-alkyl homofolates and folates are weak competitive inhibitors of 5-MeH₄-folate, but are inactive as substrates. The K_m of l-5-MeH₄-homofolate for the E. coli B enzyme (80 μm) is greater than that of 5-MeH₄-folate (35 μm), but its K_m for the Chinese hamster ovary cell transmethylase (20 μm) is less than that of 5-MeH₄-folate (35 μm). l-H₄-Homofolate is a potent competitive inhibitor (K_i = 56 nM) for the E. coli B thymidylate synthetase compared to 5-MeH₄-homofolate (K_i = 56 μM); it is also inactive as a cofactor. However, l-H₄-homofolate is a cofactor for both the HeLa S₃ and Chinese hamster ovary cell thymidylate synthetases, giving 22% of the activity observable with l-H₄-folate. Moreover, its K_m as a cofactor is only 2.1 μm compared to 17 μm for l-H₄-folate. dl-5-MeH₄-Homofolate inhibits the growth of Chinese hamster ovary cells in a reversible manner, but the inhibition is not related to the amount of B-12 holomethyltransferase in the cells.     

Purification and partial characterization of rat kidney histamine-N-methyltransferase

Histamine-N-methyltransferase (EC 2.1.1.8) was purified 1700-fold with a yield of 9% from rat kidney. Purification included ammonium sulfate precipitation, linear gradient DEAE-cellulose chromotography and S-adenosylhomocysteine affinity chromotography. The purified enzyme preparation showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 35 000. The isoelectric point of the enzyme was at pH 5.2. The purified enzyme preparation did not contain detectable amounts of histamine. The purified enzyme was totally inhibited in 100 μM parahydroxymercuric benzoate and in 10 μM iodoacetamide, and it was found to be stabilized with dithiothreitol (1 mM), suggesting that the enzyme has an SH-group in the active center. The K_m values for histamine and S-adenosylmethionine were 6.0 and 7.1 μM, respectively. 50% inhibition of histamine-N-methyltransferase was obtained at 28 μM S-adenosylhomocysteine and 100 μM methylhistamine. The purified enzyme was slightly inhibited in 1 mM methylthioadenosine. Histamine in concentrations higher than 25 μM caused substrate inhibition.     

The Endosymbiont Amoebophilus asiaticus Encodes an S-Adenosylmethionine Carrier That Compensates for Its Missing Methylation Cycle

All organisms require S-adenosylmethionine (SAM) as a methyl group donor and cofactor for various biologically important processes. However, certain obligate intracellular parasitic bacteria and also the amoeba symbiont Amoebophilus asiaticus have lost the capacity to synthesize this cofactor and hence rely on its uptake from host cells. Genome analyses revealed that A. asiaticus encodes a putative SAM transporter. The corresponding protein was functionally characterized in Escherichia coli: import studies demonstrated that it is specific for SAM and S-adenosylhomocysteine (SAH), the end product of methylation. SAM transport activity was shown to be highly dependent on the presence of a membrane potential, and by targeted analyses, we obtained direct evidence for a proton-driven SAM/SAH antiport mechanism. Sequence analyses suggest that SAM carriers from Rickettsiales might operate in a similar way, in contrast to chlamydial SAM transporters. SAM/SAH antiport is of high physiological importance, as it allows for compensation for the missing methylation cycle. The identification of a SAM transporter in A. asiaticus belonging to the Bacteroidetes phylum demonstrates that SAM transport is more widely spread than previously assumed and occurs in bacteria belonging to three different phyla (Proteobacteria, Chlamydiae, and Bacteroidetes).     

Enzymatic stereospecific preparation of fluorescent S-adenosyl-l-methionine analogs

S-Adenosyl-l-methionine (SAM) is the preferred cofactor for biological methyl group transfers to various substrates such as nucleic acids, proteins, and lipids. Here we present stereospecific (>95% of the desired enantiomer) and high-yield preparation of four fluorescent and biologically active SAM analogs and demonstrate their usefulness in binding studies. Using a fluorescence titration experiment, we obtained a K_d of 0.38 μM for the S-2,6-diaminopurinylmethionine-SAM-III riboswitch complex.