Purification and characterization of Bacillus subtilis methyl-accepting chemotaxis protein methyltransferase II

A Bacillus subtilis methyltransferase capable of methylating membrane-bound methyl-accepting chemotaxis proteins (MCPs) of a chemotaxis mutant was purified to homogeneity. MCPs are normally unmethylated in this strain. Results of gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate that the enzyme is a 30,000 molecular weight monomer. The enzyme transfers methyl groups from S-adenosylmethionine to glutamate residues of the substrates. The enzyme is activated by divalent cations and has a Km for S-adenosylmethionine of about 5 microM. It is competitively inhibited by S-adenosylhomocysteine, with a Ki of about 0.2 microM, and exhibits an in vitro assay pH optimum of 6.9. This methyltransferase is very different from another methyltransferase from B. subtilis, described previously (Ullah, A. H. J., and Ordal, G. W. (1981) Biochem. J. 199, 795-805).     

In vivo and in vitro chemotactic methylation in Bacillus subtilis

Two doublets of Bacillus subtilis membrane proteins with molecular weights of 69,000 and 71,000 and of 30,000 and 30,800, were labeled by C3H3 transfer in the absence of protein synthesis. In addition, there was intense methylation of several low-molecular-weight substances. Both doublets were missing in a chemotaxis mutant. The equivalent proteins in Escherichia coli and Salmonella typhimurium are believed to be the methyl-accepting chemotaxis proteins. The higher-molecular-weight doublet bands were increased in degree of methylation upon addition of attractant to the bacteria. A methyltransferase from B. subtilis that methylates the wild-type membrane significantly better than the mutant membrane, using S-adenosylmethionine, has been partly purified. The methylated product was alkali labile and is probably a gamma-glutamyl methyl ester, as in E. coli and S. typhimurium. Ca2+ ion inhibited the methyltransferase, with a Ki of about 80 nM. Analysis of the in vitro methylation product showed labeling of the 69,000-dalton methyl-accepting chemotaxis protein and a low-molecular-weight protein, using wild-type membrane. Labeling of the low-molecular-weight protein but not of the 69,000 dalton protein was observed when the mutant membrane was used. The chemotaxis mutant tumbled much longer than the wild type when diluted away from attractant.     

The Pseudomonas aeruginosa chemotaxis methyltransferase CheR1 impacts on bacterial surface sampling

The characterization of factors contributing to the formation and development of surface-associated bacterial communities known as biofilms has become an area of intense interest since biofilms have a major impact on human health, the environment and industry. Various studies have demonstrated that motility, including swimming, swarming and twitching, seems to play an important role in the surface colonization and establishment of structured biofilms. Thereby, the impact of chemotaxis on biofilm formation has been less intensively studied. Pseudomonas aeruginosa has a very complex chemosensory system with two Che systems implicated in flagella-mediated motility. In this study, we demonstrate that the chemotaxis protein CheR1 is a methyltransferase that binds S-adenosylmethionine and transfers a methyl group from this methyl donor to the chemoreceptor PctA, an activity which can be stimulated by the attractant serine but not by glutamine. We furthermore demonstrate that CheR1 does not only play a role in flagella-mediated chemotaxis but that its activity is essential for the formation and maintenance of bacterial biofilm structures. We propose a model in which motility and chemotaxis impact on initial attachment processes, dispersion and reattachment and increase the efficiency and frequency of surface sampling in P. aeruginosa.     

Purification and characterization of methyl-accepting chemotaxis protein methyltransferase I in Bacillus subtilis.

A methyltransferase that methylates one of the proteins involved in chemotactic adaptation to sensory stimuli in Bacillus subtilis was purified to homogeneity. The enzyme utilizes S-adenosylmethionine as donor for a methyl group that is transferred to a glutamate residue in a 69 000-mol.wt. membrane protein and also to a protein of 19 000 mol.wt. The molecular weights of the denatured enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and of the native enzyme by gel-filtration chromatography both show the protein to be a 44 000-mol.wt. monomer. Isoelectric focusing of the purified methyltransferase showed the protein to be a single species with isoelectric point pI 5.4. On the basis of a molecular weight of 44 000, the molar absorption coefficient at 262 nm of the enzyme is 10.9 x 10(4) M-1 . cm-1. The Km of the enzyme for S-adenosylmethionine is about 2 microM. The Ki for S-adenosylhomocysteine is about 0.2 microM. Ca2+ is a competitive inhibitor of methylation, with a Ki of 0.065 microM. The enzyme methylates membranes from the wild-type more efficiently than membranes isolated from a mutant strain defective in chemotaxis. The enzyme is unable to methylate Escherichia coli membranes.     

S-adenosyl-L-methionine:thioether S-methyltransferase, a new enzyme in sulfur and selenium metabolism

The final urinary excretion product of selenium detoxification is trimethylselenonium ion. An assay has been developed for the enzyme, S-adenosylmethionine:thioether S-methyltransferase, responsible for this final methylation reaction. This assay employed high pressure liquid chromatography separation and quantitation of the trimethylselenonium ion produced by thioether methyltransferase acting on S-adenosylmethionine and dimethyl selenide. The enzyme was shown to reside primarily in the cytosol of mouse lung (30 pmol/mg protein/min) and liver (7 pmol/mg protein/min). Purification from mouse lung to a preparation that exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was achieved by DEAE, gel filtration, and chromatofocusing chromatographies. Thioether methyltransferase is monomeric with a molecular weight of 28,000 and has a pI of 5.3. The pH optimum was 6.3, and Km values for dimethyl selenide and S-adenosylmethionine were 0.4 and 1.0 microM, respectively. The enzyme was inhibited 50% by 25 microM sinefungin, an analog of S-adenosylmethionine, or 40 microM S-adenosylhomocysteine, the reaction product. Pure thioether methyltransferase methylated selenium in dimethyl selenide, tellurium in dimethyl telluride, and S in dimethyl sulfide and many other thioethers. These data suggest a general role for this novel enzyme in the synthesis of onium compounds with increased aqueous solubility helpful in their excretion.     

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.     

Novel methyl transfer during chemotaxis in Bacillus subtilis

If Bacillus subtilis is incubated in radioactive methionine in the absence of protein synthesis, the methyl-accepting chemotaxis proteins (MCPs) become radioactively methylated. If the bacteria are further incubated in excess nonradioactive methionine ("cold-chased") and then given the attractant aspartate, the MCPs lose about half of their radioactivity due to turnover, in which lower specific activity methyl groups from S-adenosylmethionine (AdoMet) replace higher specific activity ones. Due to the cold-chase, the specific activity of the AdoMet pool is reduced at least 2-fold. If, later, the attractant is removed, higher specific activity methyl groups return to the MCPs. Thus, there must exist an unidentified methyl carrier that can "reversibly" receive methyl groups from the MCPs. In a similar experiment, labeled cells were transferred to a flow cell and exposed to addition and removal of attractant and of repellent. All four kinds of stimuli were found to cause methanol production. Bacteria with maximally labeled MCPs were exposed to many cycles of addition and removal of attractant; the maximum amount of radioactive methanol was evolved on the third, not the first, cycle. This result suggests that there is a precursor-product relationship between methyl groups on the MCPs and on the unidentified carrier, which might be the direct source of methanol. However, since no methanol was produced when a methyltransferase mutant, whose MCPs were unmethylated, was exposed to addition and removal of attractant or repellent, the methanol must ultimately derive from methylated MCPs.     

Effects of a methyl-deficient diet on rat liver phosphatidylcholine biosynthesis

To produce a severe choline-methionine deficiency, a synthetic L-amino acid diet, free of choline, methionine, vitamin B12, and folic acid and supplemented with guanidoacetic acid, a methyl group acceptor, was fed to female rats for 2 weeks. The in vitro activity of liver microsomal phosphatidylethanolamine methyltransferase was stimulated twofold when compared with basal diet controls. The activity of choline phosphotransferase was depressed by 86%; thus, the contribution of the methyltransferase in the overall synthesis of phosphatidylcholine apparently increased. However, measurement of the in vivo methylation of phosphatidylethanolamine by incorporation of [1,2-14C]ethanolamine into phosphatidylcholine indicates that the methylation pathway is markedly depressed in methyl deficiency. Hepatic concentrations of the methyltransferase substrate, S-adenosylmethionine, and the inhibitory metabolite, S-adenosylhomocysteine, were significantly altered such that an unfavorable environment for methylation was present in the deficient animal. The ratio of substrate to inhibitor was depressed from 5.2:1 in the controls to 1.7:1 in the livers of methyl-depleted rats. Control of transmethylation in accordance with the availability of substrates, phosphatidylethanolamine, or S-adenosylmethionine, and the level of S-adenosylhomocysteine is discussed.     

Defining the role of phosphomethylethanolamine N-methyltransferase from Caenorhabditis elegans in phosphocholine biosynthesis by biochemical and kinetic analysis

In plants and Plasmodium falciparum, the synthesis of phosphatidylcholine requires the conversion of phosphoethanolamine to phosphocholine by phosphoethanolamine methyltransferase (PEAMT). This pathway differs from the metabolic route of phosphatidylcholine synthesis used in mammals and, on the basis of bioinformatics, was postulated to function in the nematode Caenorhabditis elegans. Here we describe the cloning and biochemical characterization of a PEAMT from C. elegans (gene, pmt-2; protein, PMT-2). Although similar in size to the PEAMT from plants, which contain two tandem methyltransferase domains, PMT-2 retains only the C-terminal methyltransferase domain. RNA-mediated interference experiments in C. elegans show that PMT-2 is essential for worm viability and that choline supplementation rescues the RNAi-generated phenotype. Unlike the plant and Plasmodium PEAMT, which catalyze all three methylations in the pathway, PMT-2 catalyzes only the last two steps in the pathway, i.e., the methylation of phosphomonomethylethanolamine (P-MME) to phosphodimethylethanolamine (P-DME) and of P-DME to phosphocholine. Analysis of initial velocity patterns suggests a random sequential kinetic mechanism for PMT-2. Product inhibition by S-adenosylhomocysteine was competitive versus S-adenosylmethionine and noncompetitive versus P-DME, consistent with formation of a dead-end complex. Inhibition by phosphocholine was competitive versus each substrate. Fluorescence titrations show that all substrates and products bind to the free enzyme. The biochemical data are consistent with a random sequential kinetic mechanism for PMT-2. This work provides a kinetic basis for additional studies on the reaction mechanism of PEAMT. Our results indicate that nematodes also use the PEAMT pathway for phosphatidylcholine biosynthesis. If the essential role of PMT-2 in C. elegans is conserved in parasitic nematodes of mammals and plants, then inhibition of the PEAMT pathway may be a viable approach for targeting these parasites with compounds of medicinal or agronomic value.     

Assembly of EcoKI DNA methyltransferase requires the C-terminal region of the HsdM modification subunit

The methyltransferase component of type I DNA restriction and modification systems comprises three subunits, one DNA sequence specificity subunit and two DNA modification subunits. Limited proteolysis of the EcoKI methyltransferase shows that a 55-kDa N-terminal fragment of the 59-kDa modification subunit is resistant to degradation. We have purified this fragment and determined by mass spectrometry that proteolysis removes 43 or 44 amino acids from the C-terminus. The fragment fails to interact with the other subunits even though it still possesses secondary and tertiary structure and the ability to bind the S-adenosylmethionine cofactor. We conclude that the C-terminal region of the modification subunit of EcoKI is essential for the assembly of the EcoKI methyltransferase.     

Structure-based experimental confirmation of biochemical function to a methyltransferase,MJ0882, from hyperthermophile Methanococcus jannaschii

We have determined the three-dimensional (3-D) structure of protein MJ0882, which derives from a hypothetical open reading frame in the genome of the hyperthermophile Methanococcus jannaschii. The 3-D fold of MJ0882 at 1.8 Å highly resembles that of a methyltransferase, despite limited sequence similarity to any confirmed methyltransferase. The structure has an S-adenosylmethionine (AdoMet) binding pocket surrounded by motifs with similarities to those commonly found among AdoMet binding proteins. Preliminary biochemical experiments show that MJ0882 specifically binds to AdoMet, which is the essential co-factor for methyltransferases.     

Reverse methionine biosynthesis from S-adenosylmethionine in eukaryotic cells

The intracellular ratio between methionine and its activated form S-adenosylmethionine (AdoMet) is of crucial importance for the one-carbon metabolism. AdoMet recycling into methionine was believed to be largely achieved through the methyl and the thiomethyladenosine cycles. We show here that in yeast, AdoMet recycling actually occurs mainly through the direct AdoMet-dependent remethylation of homocysteine. Compelling evidences supporting this result were obtained owing to the identification and functional characterization of two new genes, SAM4 and MHT1, that encode the yeast AdoMet-homocysteine methyltransferase and S-methylmethionine-homocysteine methyltransferase, respectively. Homologs of the Sam4 and Mht1 proteins exist in other eucaryotes, indicating that such enzymes would be universal and not restricted to the bacterial or fungal kingdoms. New pathways for AdoMet or S-methylmethionine-dependent methionine synthesis are presented.     

Biosynthesis of caffeine by tea-leaf extracts. Enzymic formation of theobromine from 7-methylxanthine and of caffeine from theobromine.

1. Extracts prepared from tea leaves with Polyclar AT (insoluble polyvinylpyrrolidine) contained two methyltransferase activities catalysing the transfer of methyl groups from S-adenosylmethionine to 7-methylxanthine, producing theobromine, and to theobromine, producing caffeine. 2. The methyltransferases exhibited the same pH optimum (8.4) and a similar pattern of effects by metal ions, thiol inhibitors and metal-chelating reagents, both for theobromine and caffeine synthesis. Mg2+, Mn2+ and Ca2+ slightly stimulated enzyme activity but they were not essential. Paraxanthine was shown to be most active among methylxanthines, as the methyl acceptor. However, the formation of paraxanthine from 1-methylxanthine was very low and that from 7-methylxanthine was nil, suggesting that the synthesis of caffeine from paraxanthine is of little importance in intact plants. Xanthine, xanthosine, XMP and hypoxanthine were all inactive as methyl acceptors, whereas [2(-14)C]xanthine and [8(-14)C]hypoxanthine were catabolized to allantoin and urea by tea-leaf extracts. The apparent Km values are as follows: 7-methylxanthine, 1.0 times 10(-14)M; theobromine, 1.0 times 10(-3)M; paraxanthine, 0.2 times 10(-3)M; S-adenosylmethionine, 0.25 times 10(-4)M (with each of the three substrates). 3. The results suggest that the pathway for caffeine biosynthesis is as follows: 7-methylxanthine leads to theobromine leads to caffeine. In contrast, it is suggested that theophylline is synthesized from 1-methylxanthine. The methyl groups of the purine ring of caffeine are all derived directly from the methyl group of S-adenosylmethionine. Little is known about the pathways leading to the formation of 7-methylxanthine. 4. A good correlation between caffeine synthesis and shoot formation or growth of tea seedlings was shown, suggesting that the methylating systems in caffeine synthesis are closely associated with purine nucleotide and nucleic acid metabolism in tea plants.     

S-adenosylmethionine may not be essential for signal transduction during bacterial chemotaxis.

We previously showed that a mutant strain of Salmonella typhimurium completely deficient in both the chemoreceptor methylating (CheR) and demethylating (CheB) enzymes can still exhibit chemotaxis to aspartate and other attractants (J. Stock, A. Borczuk, F. Chiou, and J. E. B. Burchenal, Proc. Natl. Acad. Sci. USA 82:8364-8368, 1985). We used this cheR cheB mutant to examine the possibility of an additional requirement for S-adenosylmethionine in chemotaxis besides its role in chemoreceptor methylation. A metE mutation was transduced into a cheR cheB double mutant, and the cells were starved for methionine. Despite the fact that intracellular S-adenosylmethionine dropped from approximately 100 microM to less than 0.2 microM, chemotaxis was largely unaffected. In contrast, a corresponding cheR+ cheB+ metE mutant completely lost its chemotaxis ability after being starved for methionine. We conclude from this observation that the primary requirement for S-adenosylmethionine during bacterial chemotaxis is in the methylation of receptor proteins.     

Bovine lens gamma-glutamylcysteine synthetase. Inhibition by glutathione and adenine nucleotides

Steady-state kinetic analysis shows that glutathione binds reversibly to both Mg . enzyme and Mg . enzyme . L-glutamate forms of gamma-glutamylcysteine synthetase to form inactive complexes. The Ki values for binding to these two species of enzyme are 4 mM and 0.4 mM, respectively; those for S-methyl glutathione are 16 mM and 0.5 mM, respectively. These data suggest that glutathione is an important feedback inhibitor and contributes to the regulation of glutathione synthesis by modulating the synthesis rate of the precursor dipeptide. Adenosine 5'-diphosphate (5'ADP) is also an inhibitor and competes with both ATP and L-beta-chloroalanine for Mg . enzyme . L-glutamate and Mg . enzyme . L-glutamylphosphate, respectively. Under physiological conditions in the lens, 5' ADP competes effectively with L-cysteine for Mg . enzyme . L-glutamylphosphate, owing to the low concentration of L-cysteine, and less effectively with ATP for Mg . enzyme . L-glutamate, because of a high concentration of ATP.     

A methyl-accepting protein involved in multiple-sugar chemotaxis by Cellulomonas gelida.

Tethered-cell and capillary assays indicated that L-methionine is required by Cellulomonas gelida for its normal cell motility pattern and chemotaxis and that S-adenosylmethionine is involved in sugar chemotaxis by this cellulolytic bacterium. In addition, in vivo methylation assays showed that several proteins were methylated in the absence of protein synthesis. The incorporated methyl groups were alkali sensitive. Of special interest was the observation that the methylation level of a 51,000-Mr protein increased two- to fivefold upon addition of various sugar attractants and decreased after the removal of the attractants. The increase was less pronounced in mutants defective in sugar chemotaxis and appeared to be specifically involved with sugar chemotaxis. Furthermore, cell fractionation and in vitro methylation assays demonstrated that the 51,000-Mr protein is located in the cytoplasmic membrane. These results suggest that a specific methyl-accepting chemotaxis protein is involved in multiple-sugar chemotaxis by C gelida. During chemotaxis, the changes of methylesterase activity in C gelida cells were similar to those in Escherichia coli RP437 cells, as determined by a continuous-flow assay for methanol evolution. Thus, the mechanism of methyl-accepting chemotaxis protein-mediated chemotaxis of the gram-positive C. gelida appears to be similar to that of the gram-negative E. coli rather than to that of other gram-positive bacteria, such as Bacillus subtilis.     

Rat liver glycine methyltransferase. Cooperative binding of S-adenosylmethionine and loss of cooperativity by removal of a short NH2-terminal segment

Rat liver glycine methyltransferase, a homotetramer, exhibits sigmoidal rate behavior with respect to S-adenosylmethionine (Ogawa, H., and Fujioka, M. (1982) J. Biol. Chem. 257, 3447-3452). The binding experiment shows that the sigmoidicity observed in initial velocity kinetics is explained by the cooperative binding of S-adenosylmethionine to the catalytic sites residing on each subunit. Limited proteolysis of glycine methyltransferase with trypsin in the presence of S-adenosylmethionine yields an enzyme lacking the NH2-terminal 8 residues. The proteolytically modified enzyme retains a tetrameric structure. The truncated enzyme shows no cooperativity with respect to S-adenosylmethionine binding and kinetics. It has values of Vmax and Km for glycine identical to those of the native enzyme, but a 3-fold lower [S]0.5 value for S-adenosylmethionine. The proteolytic modification is without effect on the circular dichroism and fluorescence spectra. Furthermore, the protein fluorescence of the modified enzyme is quenched upon addition of S-adenosylmethionine to the same extent as observed with the native enzyme. These results suggest that a short NH2-terminal segment, which lies outside the active site, is important for communication between subunits.     

Studies on the activity ofKluyveromyces lactis S-adenosylmethionine: Δ 24-sterol methyltransferase in presence of polyenic antifungal agents

The S-adenosylmethionine: Δ 24-sterol methyltransferase (24 SMT) primarily considered as a mitochondrial enzyme, was recently mainly detected in lipid particles of yeasts. It catalyses the methylation of zymosterol which is an essential reaction for the synthesis of ergosterol. We have investigated in cellular extracts of twoKluyveromyces lactis strains the action of polyenic antifungal agents on the activity of this enzyme. Low concentrations of amphotericin B, candicidin and pimaricin strongly stimulate this activity, while high concentrations inhibit it or have no effect. Whatever the doses used, nystatin and filipin had no significant influence on this activity. According to the molar ratio amphotericin B/total sterols of the enzyme preparation, the interference of amphotericin B on the 24 SMT activity may result of two mechanisms.     

Optimal conditions for the use of protein L-isoaspartyl methyltransferase in assessing the isoaspartate content of peptides and proteins.

Protein L-isoaspartyl methyltransferase provides a basis for enzymatic measurement of atypical, isoaspartyl linkages which make a major contribution to protein microheterogeneity. The low Vmax of the methyltransferase reaction and the instability of the methyl ester can hinder accurate determinations, and different laboratories using different conditions have achieved discrepant values for the isoaspartate content of the same proteins. To investigate the effects of these conditions, and to optimize the assay, isoaspartyl delta sleep-inducing peptide was methylated under a variety of conditions. We found that 1 microM methyltransferase was required to obtain stoichiometric modification of 2 microM peptide in 40-min reactions at pH 6.2 and 30 degrees C. A computer model utilizing kinetic constants obtained from studies on initial rates of methylation predicted the same requirement for enzyme concentration. Carrier protein was necessary for optimal methyltransferase activity at enzyme concentrations below 0.4 microM. Stoichiometric methylation required concentrations of S-adenosylmethionine to be in substantial excess over those of peptide; 50 microM S-adenosylmethionine is the minimum needed for complete modification of 10 microM peptide. Spontaneous demethylation was significant under all conditions tested, so that the methyl ester itself never reached a ratio of 1 mol/mol of total peptide. These results demonstrate that the most accurate measurements of isoaspartate are obtained when reactions are carried out at low peptide concentrations, high S-adenosylmethionine concentrations, and high enzyme concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression and positioning of chemotaxis methylation proteins in Caulobacter

Proteins involved in chemotaxis methylation reactions have been identified in Caulobacter crescentus and their activities, times of synthesis and cellular positions have been determined. The methyl-accepting chemotaxis proteins, the methyl-transferase and the methylesterase were all shown to be active in the flagella-bearing swarmer cell, but all three activities were lost after the swarmer cells shed their flagellum and differentiated into a stalked cell. The membrane methyl-accepting chemotaxis proteins were shown to be synthesized before cell division, coincident with the synthesis of the components of the flagellum, and to be specifically localized in the membrane of the incipient swarmer cell portion of the predivisional cell. The cytoplasmic methylesterase was also found to be differentially synthesized coincident with the period of flagellar biogenesis. Furthermore, methyltransferase activity, present in the predivisional cell, was detected only in the swarmer cell upon cell division. These results demonstrate that the chemotaxis methylation machinery is positionally biased toward one portion of the predivisional cell, and that the time of expression of a set of fla and che genes is correlated with the positioning of their gene products within the cell.