Kinetic and pH studies on human phenylethanolamine N-methyltransferase

Phenylethanolamine N-methyltransferase (PNMT) catalyzes the conversion of norepinephrine (noradrenaline) to epinephrine (adrenaline) while, concomitantly, S-adenosyl-l-methionine (AdoMet) is converted to S-adenosyl-l-homocysteine. This reaction represents the terminal step in catecholamine biosynthesis and inhibitors of PNMT have been investigated, inter alia, as potential antihypertensive agents. At various times the kinetic mechanism of PNMT has been reported to operate by a random mechanism, an ordered mechanism in which norepinephrine binds first, and an ordered mechanism in which AdoMet binds first. Here we report the results of initial velocity studies on human PNMT in the absence and presence of product and dead end inhibitors. These, coupled with isothermal titration calorimetry and fluorescence binding experiments, clearly shown that hPNMT operates by an ordered sequential mechanism in which AdoMet binds first. Although the log V pH-profile was not well defined, plots of log V/K versus pH for AdoMet and phenylethanolamine, as well as the pK_i versus pH for the inhibitor, SK&F 29661, were all bell-shaped indicating that a protonated and an unprotonated group are required for catalysis.     

The activity of adenosyl-d-methionine and adenosyl-2-methylmethionine in transmethylations

The d-methionine- and 2-methyl-dl-methionine analogs of the enzymatic methyl donor, (?)S-adenosyl-l-methionine, were synthesized by methylation of S-adenosyl-d-homocysteine and S-adenosyl-2-methyl-dl-homocysteine with methyl iodide. By chromatographic purification, S-adenosyl-d-methionine and S-adenosyl-2-methyl-dl-methionine were obtained. The structure of the latter was ascertained by hydrolysis to 2-methylmethionine in strong acid, and to 5′-methylthioadenosine and 2-methylhomoserine at pH 4. Reference material of the latter compound was obtained by alkaline hydrolysis of 2-methylmethionine methylsulfonium iodide. The sulfonium compounds were tested as methyl donors with N-acetylserotonin O-methyltransferase, l-homocysteine S-methyltransferase, histamine N-methyltransferase, and guanidinoacetate N-methyltransferase. In most instances, methyl donor activity was observed.     

Structural Insights into the Catalytic Mechanism of Synechocystis Magnesium Protoporphyrin IX O-Methyltransferase (ChlM)

Magnesium protoporphyrin IX O-methyltransferase (ChlM) catalyzes transfer of the methyl group from S-adenosylmethionine to the carboxyl group of the C13 propionate side chain of magnesium protoporphyrin IX. This reaction is the second committed step in chlorophyll biosynthesis from protoporphyrin IX. Here we report the crystal structures of ChlM from the cyanobacterium Synechocystis sp. PCC 6803 in complex with S-adenosylmethionine and S-adenosylhomocysteine at resolutions of 1.6 and 1.7 Å, respectively. The structures illustrate the molecular basis for cofactor and substrate binding and suggest that conformational changes of the two “arm” regions may modulate binding and release of substrates/products to and from the active site. Tyr-28 and His-139 were identified to play essential roles for methyl transfer reaction but are not indispensable for cofactor/substrate binding. Based on these structural and functional findings, a catalytic model is proposed.     

Characterization of Glycine Sarcosine N-Methyltransferase and Sarcosine Dimethylglycine N-Methyltransferase

Glycine betaine is accumulated in cells living in high salt concentrations to balance the osmotic pressure. Glycine sarcosine N-methyltransferase (GSMT) and sarcosine dimethylglycine N-methyltransferase (SDMT) of Ectothiorhodospira halochloris catalyze the threefold methylation of glycine to betaine, with S-adenosylmethionine acting as the methyl group donor. These methyltransferases were expressed in Escherichia coli and purified, and some of their enzymatic properties were characterized. Both enzymes had high substrate specificities and pH optima near the physiological pH. No evidence of cofactors was found. The enzymes showed Michaelis-Menten kinetics for their substrates. The apparent K_m and V_max values were determined for all substrates when the other substrate was present in saturating concentrations. Both enzymes were strongly inhibited by the reaction product S-adenosylhomocysteine. Betaine inhibited the methylation reactions only at high concentrations.     

Structural insights into the histidine trimethylation activity of EgtD from Mycobacterium smegmatis

EgtD is an S-adenosyl-l-methionine (SAM)-dependent histidine N,N,N-methyltransferase that catalyzes the formation of hercynine from histidine in the ergothioneine biosynthetic process of Mycobacterium smegmatis. Ergothioneine is a secreted antioxidant that protects mycobacterium from oxidative stress. Here, we present three crystal structures of EgtD in the apo form, the histidine-bound form, and the S-adenosyl-l-homocysteine (SAH)/histidine-bound form. The study revealed that EgtD consists of two distinct domains: a typical methyltransferase domain and a unique substrate binding domain. The histidine binding pocket of the substrate binding domain primarily recognizes the imidazole ring and carboxylate group of histidine rather than the amino group, explaining the high selectivity for histidine and/or (mono-, di-) methylated histidine as substrates. In addition, SAM binding to the MTase domain induced a conformational change in EgtD to facilitate the methyl transfer reaction. The structural analysis provides insights into the putative catalytic mechanism of EgtD in a processive trimethylation reaction.     

An Alternative Mechanism for the Methylation of Phosphoethanolamine Catalyzed by Plasmodium falciparum Phosphoethanolamine Methyltransferase

The phosphobase methylation pathway catalyzed by the phosphoethanolamine methyltransferase in Plasmodium falciparum (PfPMT), the malaria parasite, offers an attractive target for anti-parasitic drug development. PfPMT methylates phosphoethanolamine (pEA) to phosphocholine for use in membrane biogenesis. Quantum mechanics and molecular mechanics (QM/MM) calculations tested the proposed reaction mechanism for methylation of pEA involving the previously identified Tyr-19–His-132 dyad, which indicated an energetically unfavorable mechanism. Instead, the QM/MM calculations suggested an alternative mechanism involving Asp-128. The reaction coordinate involves the stepwise transfer of a proton to Asp-128 via a bridging water molecule followed by a typical S_n2-type methyl transfer from S-adenosylmethionine to pEA. Functional analysis of the D128A, D128E, D128Q, and D128N PfPMT mutants shows a loss of activity with pEA but not with the final substrate of the methylation pathway. X-ray crystal structures of the PfPMT-D128A mutant in complex with S-adenosylhomocysteine and either pEA or phosphocholine reveal how mutation of Asp-128 disrupts a hydrogen bond network in the active site. The combined QM/MM, biochemical, and structural studies identify a key role for Asp-128 in the initial step of the phosphobase methylation pathway in Plasmodium and provide molecular insight on the evolution of multiple activities in the active site of the PMT.     

Thiol S-methyltransferase from rat liver.

The thiol S-methyltransferase from rat liver has been solubilized and prepared in homogeneous form. The enzyme exists in a monomer of M_r 28,000 although enzyme activity is highly unstable with a half-life of 4 days under the best conditions of storage. The reaction requires S-adenosylmethionine as methyl donor but, as is the case with many enzymes active in detoxification, a large variety of lipophilic compounds can serve as acceptors. Acceptor activity is limited to thiols. The naturally occurring hydrophilic thiols, glutathione and cysteine, act neither as substrates nor as inhibitors. The course of the reaction is biphasic with an initial rapid formation of product that is followed by a slower linear rate. The suggestion is offered that this behavior reflects the slow dissociation of an enzyme-product complex composed of enzyme and S-adenosyl-homocysteine.     

Structure and Biophysical Characterization of the S-Adenosylmethionine-dependent O-Methyltransferase PaMTH1, a Putative Enzyme Accumulating during Senescence of Podospora anserina

Low levels of reactive oxygen species (ROS) act as important signaling molecules, but in excess they can damage biomolecules. ROS regulation is therefore of key importance. Several polyphenols in general and flavonoids in particular have the potential to generate hydroxyl radicals, the most hazardous among all ROS. However, the generation of a hydroxyl radical and subsequent ROS formation can be prevented by methylation of the hydroxyl group of the flavonoids. O-Methylation is performed by O-methyltransferases, members of the S-adenosyl-l-methionine (SAM)-dependent O-methyltransferase superfamily involved in the secondary metabolism of many species across all kingdoms. In the filamentous fungus Podospora anserina, a well established aging model, the O-methyltransferase (PaMTH1) was reported to accumulate in total and mitochondrial protein extracts during aging. In vitro functional studies revealed flavonoids and in particular myricetin as its potential substrate. The molecular architecture of PaMTH1 and the mechanism of the methyl transfer reaction remain unknown. Here, we report the crystal structures of PaMTH1 apoenzyme, PaMTH1-SAM (co-factor), and PaMTH1-S-adenosyl homocysteine (by-product) co-complexes refined to 2.0, 1.9, and 1.9 Å, respectively. PaMTH1 forms a tight dimer through swapping of the N termini. Each monomer adopts the Rossmann fold typical for many SAM-binding methyltransferases. Structural comparisons between different O-methyltransferases reveal a strikingly similar co-factor binding pocket but differences in the substrate binding pocket, indicating specific molecular determinants required for substrate selection. Furthermore, using NMR, mass spectrometry, and site-directed active site mutagenesis, we show that PaMTH1 catalyzes the transfer of the methyl group from SAM to one hydroxyl group of the myricetin in a cation-dependent manner.     

Biosynthesis of a retrochalcone,echinatin: Involvement of O-methyltransferase to licodione

In order to clarify the O-methylation step in the biosynthesis of a retrochalcone, echinatin(4,4′-dihydroxy-2-methoxychalcone), methyl transfer from S-adenosyl-l-methionine (SAM) to licodione (1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)-1,3-propanedione) in the cell-free extract of the cultured cells of Glycyrrhiza echinata was examined. Time course of methyl transferring activity during culture cycle in 4 strains was correlated to echinatin content. The enzyme catalysing this reaction, licodione O-methyltransferase (LMT), was purified 135-fold. Substrate specificity studies implied that the hydroxy group ortho to the C₃ linkage in licodione was methylated in this reaction. O-Methyl-licodiones were synthesized for comparison and the sole product of LMT-catalysed reaction was identified as 2′-O-methyl-licodione. A possible scheme for the last steps of echinatin biosynthesis is proposed.     

Partial Purification and Characterization of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit epsilonN-Methyltransferase

The large subunit (LS) of tobacco (Nicotiana rustica) ribulose-1,5-bisphosphate carboxylase/oxygenase (ribulose-P₂ carboxylase) contains a trimethyllysyl residue at position 14, whereas this position is unmodified in spinach ribulose-P₂ carboxylase. A protein fraction was isolated from tobacco chloroplasts by rate-zonal centrifugation and anion-exchange fast protein liquid chromatography that catalyzed transfer of methyl groups from S-adenosyl-[methyl-³H]-l-methionine to spinach ribulose-P₂ carboxylase. ³H-Methyl groups incorporated into spinach ribulose-P₂ carboxylase were alkaline stable but could be removed by limited tryptic proteolysis. Reverse-phase high-performance liquid chromatography of the tryptic peptides released after proteolysis showed that the penultimate N-terminal peptide from the LS of spinach ribulose-P₂ carboxylase contained the site of methylation, which was identified as lysine-14. Thus, the methyltransferase activity can be attributed to S-adenosylmethionine:ribulose-P₂ carboxylase LS (lysine) `N-methyltransferase, a previously undescribed chloroplast enzyme. The partially purified enzyme was specific for ribulose-P₂ carboxylase and exhibited apparent K_m values of 10 micromolar for S-adenosyl-l-methionine and 18 micromolar for ribulose-P₂ carboxylase, a V_max of 700 picomoles CH₃ groups transferred per minute per milligram protein, and a broad pH optimum from 8.5 to 10.0. S-Adenosylmethionine:ribulose-P₂ carboxylase LS (lysine)^εN-methyltransferase was capable of incorporating 24 ³H-methyl groups per spinach ribulose-P₂ carboxylase holoenzyme, forming 1 mole of trimethyllysine per mole of ribulose-P₂ carboxylase LS, but was inactive on ribulose-P₂ carboxylases that contain a trimethyllysyl residue at position 14 in the LS. The enzyme did not distinguish between activated (Mg²⁺ and CO₂) and unactivated forms of ribulose-P₂ carboxylase as substrates. However, complexes of activated ribulose-P₂ carboxylase with the reaction-intermediate analogue 2′-carboxy-d-arabinitol-1,5-bisphosphate, or unactivated spinach ribulose-P₂ carboxylase with ribulose-1,5-bisphosphate, were poor substrates for tobacco LS ^εN-methyltransferase.     

Glycine N-Methyltransferase and Regulation of S-Adenosylmethionine Levels

Methylation is a major biological process. It has been shown to be important in formation of compounds such as phosphatidylcholine, creatine, and many others and also participates in epigenetic effects through methylation of histones and DNA. The donor of methyl groups for almost all cellular methylation reactions is S-adenosylmethionine. It seems that the level of S-adenosylmethionine must be regulated in response to developmental stages and metabolic changes, and the enzyme glycine N-methyltransferase has been shown to play a major role in such regulation in mammals. This minireview will focus on the latest discoveries in the elucidation of the mechanism of that regulation.     

Transfer RNA methyltransferase and glycine N-methyltransferase activity during Rana pipiens development.

Changes in the activity of the tRNA methyltransferases have been found in all differentiating systems studied. Activity was examined in extracts of Rana pipiens embryos and in larval and adult liver by in vitro assay using S-adenosyl-l-[methyl-¹⁴C]methionine as the methyl donor. Specific activities of tRNA methyltransferases decreased, beginning with the time of feeding, when using high concentrations of the crude liver enzyme. A new methyltransferase activity, glycine N-methyltransferase, appeared at the time of feeding. Apparently, the glycine methyltransferase is active before the onset of any of the characteristic metamorphic changes of other liver enzymes. Using partially purified enzyme from adult liver, the K_m of glycine methyltransferase for S-adenosylmethionine is 0.3 mM and the K_i for S-adenosylhomocysteine, a competitive inhibitor, is 0.08 mM.     

The geometry of the thioester group and its implications for the chemistry of acyl coenzyme A

L-929 cell surface membranes were incubated with S-adenosyl-l-[methyl-³H]-methionine and found to contain phosphatidylethanolamine: S-adenosylmethionine N-methyltransferase (phosphatidylethanolamine N-methyltransferase) activity. The enzyme or combination of enzymes responsible for this activity methylated endogenous phosphatidylethanolamine and its methylated derivatives to yield phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylcholine. Maximum enzyme activity was expressed at pH 6.9, the reaction was not dependent on the presence of divalent cations, and exogenously added phospholipids did not stimulate the rate of reaction. Phospholipid methylation was inhibited by S-adenosyl-l-homocysteine and by local anaesthetic drugs such as chlorpromazine and tetracaine which partition into the lipid bilayer. Control experiments demonstrated that the surface membrane-associated methyltransferase activity was not due to contamination of surface membrane preparations with intracellular membranes. Surface membranes were found to have higher specific methyltransferase activities than whole L-cell homogenates or endoplasmic reticulum-enriched microsomes. The low rate of methyltransferase function expressed in vitro (approximately 1 pmol/min · mg protein) suggests that phospholipid methylation is not a major metabolic source of surface membrane phosphatidylcholine.     

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.     

Evolution of putrescine N-methyltransferase from spermidine synthase demanded alterations in substrate binding

Putrescine N-methyltransferase (PMT) catalyses S-adenosylmethionine (SAM)-dependent methylation of putrescine in tropane alkaloid biosynthesis. PMT presumably evolved from the ubiquitous spermidine synthase (SPDS). SPDS protein structure suggested that only few amino acid exchanges in the active site were necessary to achieve PMT activity. Protein modelling, mutagenesis, and chimeric protein construction were applied to trace back evolution of PMT activity from SPDS. Ten amino acid exchanges in Datura stramonium SPDS dismissed the hypothesis of facile generation of PMT activity in existing SPDS proteins. Chimeric PMT and SPDS enzymes were active and indicated the necessity for a different putrescine binding site when PMT developed.     

Mechanisms for enzymatic reduction of nitric oxide to nitrous oxide - A comparison between nitric oxide reductase and cytochrome c oxidase

Cytochrome c oxidases (CcO) reduce O₂ to H₂O in the respiratory chain of mitochondria and many aerobic bacteria. In addition, some species of CcO can also reduce NO to N₂O and water while others cannot. Here, the mechanism for NO-reduction in CcO is investigated using quantum mechanical calculations. Comparison is made to the corresponding reaction in a “true” cytochrome c-dependent NO reductase (cNOR). The calculations show that in cNOR, where the reduction potentials are low, the toxic NO molecules are rapidly reduced, while the higher reduction potentials in CcO lead to a slower or even impossible reaction, consistent with experimental observations. In both enzymes the reaction is initiated by addition of two NO molecules to the reduced active site, forming a hyponitrite intermediate. In cNOR, N₂O can then be formed using only the active-site electrons. In contrast, in CcO, one proton-coupled reduction step most likely has to occur before N₂O can be formed, and furthermore, proton transfer is most likely rate-limiting. This can explain why different CcO species with the same heme a₃-Cu active site differ with respect to NO reduction efficiency, since they have a varying number and/or properties of proton channels. Finally, the calculations also indicate that a conserved active site valine plays a role in reducing the rate of NO reduction in CcO.     

Synthesis of methylated ethanolamine moieties: regulation by choline in soybean and carrot

The results of experiments in which intact plants of Lemna paucicostata were labeled with either l-[³H₃C]methionine, l-[¹⁴CH₃]methionine, or [1,2-¹⁴C]ethanolamine support the conclusion that growth in concentrations of choline of 3.0 micromolar or above brings about marked decreases in the rate of biosynthesis of methylated forms of ethanolamine (normally present chiefly as phosphatidylcholine, with lesser amounts of choline and phosphocholine). The in vivo locus of the block is at the committing step in the biosynthetic sequence at which phosphoethanolamine is methylated by S-adenosylmethionine to form phosphomethylethanolamine. The block is highly specific: flow of methyl groups originating in methionine continues into S-adenosylmethionine, S-methylmethionine, the methyl moieties of pectin methyl ester, and other methylated metabolites. When choline uptake is less than the total that would be synthesized by control plants, phosphoethanolamine methylation is down-regulated to balance the uptake; total plant content of choline and its derivatives remains essentially constant. At maximum down-regulation, phosphoethanolamine methylation continues at 5 to 10% of normal. A specific decrease in the total available activity of AdoMet: phosphoethanolamine N-methyltransferase, as well as feedback inhibition of this enzyme by phosphocholine, and prevention of accumulation of phosphoethanolamine by down-regulation of ethanolamine synthesis may each contribute to effective control of phosphoethanolamine methylation. This down-regulation may necessitate major changes in S-adenosylmethionine metabolism. Such changes are discussed.     

Reductive elimination pathway for homocysteine to methionine conversion in cobalamin-dependent methionine synthase

Density functional theory has been applied to investigate the methyl transfer from methylcobalamin (MeCbl) cofactor to homocysteine (Hcy) as catalyzed by methionine synthase (MetH). Specifically, the S_N2 and the reductive elimination pathways have been probed as the possible mechanistic pathways for the methyl transfer reaction. The calculations indicate that the activation barrier for the reductive elimination reaction (24.4 kcal mol⁻¹) is almost four times higher than that for the S_N2 reaction (7.3 kcal mol⁻¹). This high energy demand of the reductive elimination pathway is rooted in the structural distortion of the corrin ring that is induced en route to the formation of the triangular transition state. Furthermore, the reductive elimination reaction demands the syn accommodation of the methyl group and the substrate over the upper face of the corrin ring, which also accounts for the high energy demand of the reaction. Consequently, the reductive elimination pathway for MetH-catalyzed methyl transfer from MeCbl to Hcy cannot be considered as one of the possible mechanistic routes.     

Substrate-bound Crystal Structures Reveal Features Unique to Mycobacterium tuberculosis N-Acetyl-glucosamine 1-Phosphate Uridyltransferase and a Catalytic Mechanism for Acetyl Transfer

N-Acetyl-glucosamine-1-phosphate uridyltransferase (GlmU), a bifunctional enzyme involved in bacterial cell wall synthesis is exclusive to prokaryotes. GlmU, now recognized as a promising target to develop new antibacterial drugs, catalyzes two key reactions: acetyl transfer and uridyl transfer at two independent domains. Hitherto, we identified GlmU from Mycobacterium tuberculosis (GlmU^Mtb) to be unique in possessing a 30-residue extension at the C terminus. Here, we present the crystal structures of GlmU^Mtb in complex with substrates/products bound at the acetyltransferase active site. Analysis of these and mutational data, allow us to infer a catalytic mechanism operative in GlmU^Mtb. In this S_N2 reaction, His-374 and Asn-397 act as catalytic residues by enhancing the nucleophilicity of the attacking amino group of glucosamine 1-phosphate. Ser-416 and Trp-460 provide important interactions for substrate binding. A short helix at the C-terminal extension uniquely found in mycobacterial GlmU provides the highly conserved Trp-460 for substrate binding. Importantly, the structures reveal an uncommon mode of acetyl-CoA binding in GlmU^Mtb; we term this the U conformation, which is distinct from the L conformation seen in the available non-mycobacterial GlmU structures. Residues, likely determining U/L conformation, were identified, and their importance was evaluated. In addition, we identified that the primary site for PknB-mediated phosphorylation is Thr-418, near the acetyltransferase active site. Down-regulation of acetyltransferase activity upon Thr-418 phosphorylation is rationalized by the structures presented here. Overall, this work provides an insight into substrate recognition, catalytic mechanism for acetyl transfer, and features unique to GlmU^Mtb, which may be exploited for the development of inhibitors specific to GlmU.     

The enzymatic synthesis of S-adenosyl-l-[2(n)-H]homocysteine

A rapid, efficient method is described for the enzymatic conversion of S-adenosyl-l-[2(n)-³H]methionine to S-adenosyl-l-[2(n)-³H]homocysteine. Partially purified glycine N-methyltransferase is used in the reaction which yields 98% conversion. The product is purified using high-pressure liquid chromatography and is concentrated by lyophilization. S-Adenosyl-l-[2(n)-³H]homocysteine synthesized by this method is an active substrate for S-adenosylhomocysteine (SAH) hydrolase. A novel assay procedure for SAH hydrolase is also described, in which unreacted S-adenosyl-l-[2(n)-³H]homocysteine is removed by adsorption to dextran-coated charcoal.