Structure and reaction mechanism of phosphoethanolamine methyltransferase from the malaria parasite Plasmodium falciparum: an antiparasitic drug target

In the malarial parasite Plasmodium falciparum, a multifunctional phosphoethanolamine methyltransferase (PfPMT) catalyzes the methylation of phosphoethanolamine (pEA) to phosphocholine for membrane biogenesis. This pathway is also found in plant and nematodes, but PMT from these organisms use multiple methyltransferase domains for the S-adenosylmethionine (AdoMet) reactions. Because PfPMT is essential for normal growth and survival of Plasmodium and is not found in humans, it is an antiparasitic target. Here we describe the 1.55 Å resolution crystal structure of PfPMT in complex with AdoMet by single-wavelength anomalous dispersion phasing. In addition, 1.19–1.52 Å resolution structures of PfPMT with pEA (substrate), phosphocholine (product), sinefungin (inhibitor), and both pEA and S-adenosylhomocysteine bound were determined. These structures suggest that domain rearrangements occur upon ligand binding and provide insight on active site architecture defining the AdoMet and phosphobase binding sites. Functional characterization of 27 site-directed mutants identifies critical active site residues and suggests that Tyr-19 and His-132 form a catalytic dyad. Kinetic analysis, isothermal titration calorimetry, and protein crystallography of the Y19F and H132A mutants suggest a reaction mechanism for the PMT. Not only are Tyr-19 and His-132 required for phosphobase methylation, but they also form a “catalytic” latch that locks ligands in the active site and orders the site for catalysis. This study provides the first insight on this antiparasitic target enzyme essential for survival of the malaria parasite; however, further studies of the multidomain PMT from plants and nematodes are needed to understand the evolutionary division of metabolic function in the phosphobase pathway of these organisms.     

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.     

Phosphoethanolamine N-methyltransferase (PMT-1) catalyses the first reaction of a new pathway for phosphocholine biosynthesis in Caenorhabditis elegans

The development of nematicides targeting parasitic nematodes of animals and plants requires the identification of biochemical targets not found in host organisms. Recent studies suggest that Caenorhabditis elegans synthesizes phosphocholine through the action of PEAMT (S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferases) that convert phosphoethanolamine into phosphocholine. Here, we examine the function of a PEAMT from C. elegans (gene: pmt-1; protein: PMT-1). Our analysis shows that PMT-1 only catalyses the conversion of phosphoethanolamine into phospho-monomethylethanolamine, which is the first step in the PEAMT pathway. This is in contrast with the multifunctional PEAMT from plants and Plasmodium that perform multiple methylations in the pathway using a single enzyme. Initial velocity and product inhibition studies indicate that PMT-1 uses a random sequential kinetic mechanism and is feedback inhibited by phosphocholine. To examine the effect of abrogating PMT-1 activity in C. elegans, RNAi (RNA interference) experiments demonstrate that pmt-1 is required for worm growth and development and validate PMT-1 as a potential target for inhibition. Moreover, providing pathway metabolites downstream of PMT-1 reverses the RNAi phenotype of pmt-1. Because PMT-1 is not found in mammals, is only distantly related to the plant PEAMT and is conserved in multiple parasitic nematodes of humans, animals and crop plants, inhibitors targeting it may prove valuable in human and veterinary medicine and agriculture.     

An enzyme-coupled ultrasensitive luminescence assay for protein methyltransferases

Epigenetic regulation through protein posttranslational modifications is essential in development and disease. Among the key chemical modifications is protein methylation carried out by protein methyltransferases (PMTs). Quantitative and sensitive PMT activity assays can provide valuable tools to investigate PMT functions. Here we developed an enzyme-coupled luminescence assay for S-adenosyl-l-methionine (AdoMet/SAM)-based PMTs. In this assay, S-adenosyl-l-homocystine (AdoHcy/SAH), the by-product of PMT-involved methylation, is sequentially converted to adenine, adenosine monophosphate, and then adenosine 5′-triphosphate (ATP) by 5′-methylthio-adenosine/AdoHcy nucleosidase (MTAN), adenine phosphoribosyl transferase (APRT), and pyruvate orthophosphate dikinase (PPDK), respectively. The resultant ATP can be readily quantified with a luciferin/luciferase kit. This assay is featured for its quantitative linear response to AdoHcy and the ultrasensitivity to 0.3 pmol of AdoHcy. With this assay, the kinetic parameters of SET7/9 methylation were characterized and unambiguously support an ordered mechanism with AdoMet binding as the initial step, followed by the substrate binding and the rate-limiting methylation. The luminescence assay is also expected to be generally applicable to many other AdoMet-dependent enzymes. In addition, the mix-and-measure 96-/384-well format of our assay makes it suitable for automation and high throughput. Our enzyme-coupled luminescence assay, therefore, represents a convenient and ultrasensitive approach to examine methyltransferase activities and identify methyltransferase inhibitors.     

Direct evidence for methyl group coordination by carbon-oxygen hydrogen bonds in the lysine methyltransferase SET7/9

SET domain lysine methyltransferases (KMTs) are S-adenosylmethionine (AdoMet)-dependent enzymes that catalyze the site-specific methylation of lysyl residues in histone and non-histone proteins. Based on crystallographic and cofactor binding studies, carbon-oxygen (CH · · · O) hydrogen bonds have been proposed to coordinate the methyl groups of AdoMet and methyllysine within the SET domain active site. However, the presence of these hydrogen bonds has only been inferred due to the uncertainty of hydrogen atom positions in x-ray crystal structures. To experimentally resolve the positions of the methyl hydrogen atoms, we used NMR (1)H chemical shift coupled with quantum mechanics calculations to examine the interactions of the AdoMet methyl group in the active site of the human KMT SET7/9. Our results indicated that at least two of the three hydrogens in the AdoMet methyl group engage in CH · · · O hydrogen bonding. These findings represent direct, quantitative evidence of CH · · · O hydrogen bond formation in the SET domain active site and suggest a role for these interactions in catalysis. Furthermore, thermodynamic analysis of AdoMet binding indicated that these interactions are important for cofactor binding across SET domain enzymes.     

cDNA cloning of phosphoethanolamine N-methyltransferase from spinach by complementation in Schizosaccharomyces pombe and characterization of the recombinant enzyme

The N-methylation of phosphoethanolamine is the committing step in choline biogenesis in plants and is catalyzed by S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferase (PEAMT, EC ). A spinach PEAMT cDNA was isolated by functional complementation of a Schizosaccharomyces pombe cho2(-) mutant and was shown to encode a protein with PEAMT activity and without ethanolamine- or phosphatidylethanolamine N-methyltransferase activity. The PEAMT cDNA specifies a 494-residue polypeptide comprising two similar, tandem methyltransferase domains, implying that PEAMT arose by gene duplication and fusion. Data base searches suggested that PEAMTs with the same tandem structure are widespread among flowering plants. Size exclusion chromatography of the recombinant enzyme indicates that it exists as a monomer. PEAMT catalyzes not only the first N-methylation of phosphoethanolamine but also the two subsequent N-methylations, yielding phosphocholine. Monomethyl- and dimethylphosphoethanolamine are detected as reaction intermediates. A truncated PEAMT lacking the C-terminal methyltransferase domain catalyzes only the first methylation. Phosphocholine inhibits both the wild type and the truncated enzyme, although the latter is less sensitive. Salinization of spinach plants increases PEAMT mRNA abundance and enzyme activity in leaves by about 10-fold, consistent with the high demand in stressed plants for choline to support glycine betaine synthesis.     

Non‐specific phospholipases C,NPC2 and NPC6, are required for root growth in Arabidopsis

Mutants in lipid metabolism often show a lethal phenotype during reproduction that prevents investigating a specific role of the lipid during different developmental processes. We focused on two non‐specific phospholipases C, NPC2 and NPC6, whose double knock‐out causes a gametophyte‐lethal phenotype. To investigate the role of NPC2 and NPC6 during vegetative growth, we produced transgenic knock‐down mutant lines that circumvent the lethal effect during gametogenesis. Despite no defect observed in leaves, root growth was significantly retarded, with abnormal cellular architecture in root columella cells. Furthermore, the short root phenotype was rescued by exogenous supplementation of phosphocholine, a product of non‐specific phospholipase C (NPC) ‐catalyzed phosphatidylcholine hydrolysis. The expression of phospho‐base N‐methyltransferase 1 (PMT1), which produces phosphocholine and is required for root growth, was induced in the knock‐down mutant lines and was attenuated after phosphocholine supplementation. These results suggest that NPC2 and NPC6 may be involved in root growth by producing phosphocholine via metabolic interaction with a PMT‐catalyzed pathway, which highlights a tissue‐specific role of NPC enzymes in vegetative growth beyond the gametophyte‐lethal phenotype.     

Differentiating analogous tRNA methyltransferases by fragments of the methyl donor

Bacterial TrmD and eukaryotic-archaeal Trm5 form a pair of analogous tRNA methyltransferase that catalyze methyl transfer from S-adenosyl methionine (AdoMet) to N(1) of G37, using catalytic motifs that share no sequence or structural homology. Here we show that natural and synthetic analogs of AdoMet are unable to distinguish TrmD from Trm5. Instead, fragments of AdoMet, adenosine and methionine, are selectively inhibitory of TrmD rather than Trm5. Detailed structural information of the two enzymes in complex with adenosine reveals how Trm5 escapes targeting by adopting an altered structure, whereas TrmD is trapped by targeting due to its rigid structure that stably accommodates the fragment. Free energy analysis exposes energetic disparities between the two enzymes in how they approach the binding of AdoMet versus fragments and provides insights into the design of inhibitors selective for TrmD.     

Crystal structure of the N‐terminal methyltransferase‐like domain of anamorsin

Anamorsin is a recently identified molecule that inhibits apoptosis during hematopoiesis. It contains an N‐terminal methyltransferase‐like domain and a C‐terminal Fe‐S cluster motif. Not much is known about the function of the protein. To better understand the function of anamorsin, we have solved the crystal structure of the N‐terminal domain at 1.8 Å resolution. Although the overall structure resembles a typical S‐adenosylmethionine (SAM) dependent methyltransferase fold, it lacks one α‐helix and one β‐strand. As a result, the N‐terminal domain as well as the full‐length anamorsin did not show S‐adenosyl‐l ‐methionine (AdoMet) dependent methyltransferase activity. Structural comparisons with known AdoMet dependent methyltransferases reveals subtle differences in the SAM binding pocket that preclude the N‐terminal domain from binding to AdoMet. The N‐terminal methyltransferase‐like domain of anamorsin probably functions as a structural scaffold to inhibit methyl transfers by out‐competing other AdoMet dependant methyltransferases or acts as bait for protein–protein interactions.Proteins 2014; 82:1066–1071. © 2013 Wiley Periodicals, Inc.     

Novel structural arrangement of nematode cystathionine β-synthases: characterization of Caenorhabditis elegans CBS-1

CBSs (cystathionine β-synthases) are eukaryotic PLP (pyridoxal 5 *-phosphate)-dependent proteins that maintain cellular homocysteine homoeostasis and produce cystathionine and hydrogen sulfide. In the present study, we describe a novel structural arrangement of the CBS enzyme encoded by the cbs-1 gene of the nematode Caenorhabditis elegans. The CBS-1 protein contains a unique tandem repeat of two evolutionarily conserved catalytic regions in a single polypeptide chain. These repeats include a catalytically active C-terminal module containing a PLP-binding site and a less conserved N-terminal module that is unable to bind the PLP cofactor and cannot catalyse CBS reactions, as demonstrated by analysis of truncated variants and active-site mutant proteins. In contrast with other metazoan enzymes, CBS-1 lacks the haem and regulatory Bateman domain essential for activation by AdoMet (S-adenosylmethionine) and only forms monomers. We determined the tissue and subcellular distribution of CBS-1 and showed that cbs-1 knockdown by RNA interference leads to delayed development and to an approximately 10-fold elevation of homocysteine concentrations in nematode extracts. The present study provides the first insight into the metabolism of sulfur amino acids and hydrogen sulfide in C. elegans and shows that nematode CBSs possess a structural feature that is unique among CBS proteins.     

1H, 13C, and 15N chemical shift assignments for PfPMT, a phosphoethanolamine methyltransferase from Plasmodium falciparum

Phosphoethanolamine methyltransferases (PMTs also known as PEAMTs) catalyze the three-step s-adenosyl-methionione-dependent methylation of phosphoethanolamine to form phosphocholine. These enzymes play an important function in the synthesis of phosphatidylcholine, the major phospholipid in the membranes of lower and higher eukaryotes, as well as in the production of the compatible solute and osmoprotectant glycine betaine in plants. Genetic studies in plants, Caenhorhabditis elegans and Plasmodium falciparum have demonstrated that disruption of PMT activity results in severe defects in important cellular processes such as development, replication, survival and sexual maturation and differentiation. Here we report chemical shift assignments for PfPMT, the PMT from Plasmodium falciparum. X-ray crystal structures have been recently reported for complexes of PfPMT, but the structure of the apoenzyme remains unknown. The solution structure of the apoenzyme will help to elucidate important details of the mechanism of substrate binding by PfPMT, as residues comprising the substrate binding site are inaccessible to solvent in the conformation evident in the available crystal structures. In addition to enabling determination of the solution structure of the apoenzyme, the assignments will facilitate additional investigations into the interaction of PfPMT with its substrates and inhibitors.     

A mutant HpaII methyltransferase functions as a mutator enzyme.

DNA (cytosine-5)-methyltransferases can cause deamination of cytosine when the cofactor S-adenosylmethionine (AdoMet) is limiting and thus function as sequence-specific C-->U mutator enzymes. Here we explored whether mutations causing inactivation of the cofactor binding activity of the HpaII methyltransferase, thus mimicking conditions of limiting AdoMet concentration, could convert a DNA methyltransferase to a C-->U mutator enzyme. We created two mutator enzymes from the HpaII methyltransferase (F38S and G40D) which both showed enhanced cytosine deamination activities in vitro and in vivo. Interestingly, the G:U mispairs generated by these enzymes were not repaired completely in bacteria equipped with uracil-DNA glycosylase-initiated repair machinery, giving rise to a potent mutator phenotype. This is the first report showing the creation of mutator enzymes from a DNA methyltransferase and the demonstration of their mutagenicity in living cells.     

Synthesis of methylated ethanolamine moieties: regulation by choline in lemna

The results of experiments in which intact plants of Lemna paucicostata were labeled with either l-[(3)H(3)C]methionine, l-[(14)CH(3)]methionine, or [1,2-(14)C]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.     

Structure of a binary complex of HhaI methyltransferase with S-adenosyl-L-methionine formed in the presence of a short non-specific DNA oligonucleotide

We have determined a structure for a complex formed between HhaI methyltransferase (M.HhaI) and S-adenosyl-L-methionine (AdoMet) in the presence of a non-specific short oligonucleotide. M.HhaI binds to the non-specific short oligonucleotides in solution. Although no DNA is incorporated in the crystal, AdoMet binds in a primed orientation, identical with that observed in the ternary complex of the enzyme, cognate DNA, and AdoMet or S-adenosyl-L-homocysteine (AdoHcy). This orientation differs from the previously observed unprimed orientation in the M.HhaI-AdoMet binary complex, where the S+-CH3 unit of AdoMet is protected by a favorable cation-pi interaction with Trp41. The structure suggests that the presence of DNA can guide AdoMet into the primed orientation. These results shed new light on the proposed ordered mechanism of binding and explains the stable association between AdoMet and M.HhaI.     

Crystal structure of Streptococcus pneumoniae Sp1610, a putative tRNA methyltransferase,in complex with S‐adenosyl‐L‐methionine

Streptococcus pneumoniae Sp1610, a Class‐I fold S‐adenosylmethionine (AdoMet)‐dependent methyltransferase, is a member of the COG2384 family in the Clusters of Orthologous Groups database, which catalyzes the methylation of N¹‐adenosine at position 22 of bacterial tRNA. We determined the crystal structure of Sp1610 in the ligand‐free and the AdoMet‐bound forms at resolutions of 2.0 and 3.0 Å, respectively. The protein is organized into two structural domains: the N‐terminal catalytic domain with a Class I AdoMet‐dependent methyltransferase fold, and the C‐terminal substrate recognition domain with a novel fold of four α‐helices. Observations of the electrostatic potential surface revealed that the concave surface located near the AdoMet binding pocket was predominantly positively charged, and thus this was predicted to be an RNA binding area. Based on the results of sequence alignment and structural analysis, the putative catalytic residues responsible for substrate recognition are also proposed.     

Photoaffinity labelling of methyltransferase enzymes with S-adenosylmethionine: effects of methyl acceptor substrates

Radioactivity from 3H-[methyl]-S-adenosyl-L-methionine (AdoMet) was covalently bound to protein-O-carboxylmethyltransferase and phenylethanolamine N-methyltransferase following 10-15 min irradiation by short-wave ultraviolet light. This photoaffinity binding of 3H-[methyl]-AdoMet was blocked by S-adenosylhomocysteine and sinefungin, but was not affected by 5 mM dithiothreitol. The binding was also inhibited by including methyl acceptors such as calmodulin (protein-O-carboxylmethyltransferase) or phenylethanolamine (phenylethanolamine N-methyltransferase) in the photoaffinity incubation. Staphlococcus V8 protease digests of 3H-[methyl]-AdoMet/enzyme complexes revealed that the primary structure around the AdoMet binding site is different in these two enzymes. Thus, protein-O-carboxylmethyltransferase, a large molecule methyltransferase, can covalently bind 3H-[methyl]-AdoMet in a manner similar to that of phenylethanolamine-N-methyltransferase.     

Auto-methylation of the mouse DNA-(cytosine C5)-methyltransferase Dnmt3a at its active site cysteine residue

The Dnmt3a DNA methyltransferase is responsible for establishing DNA methylation patterns during mammalian development. We show here that the mouse Dnmt3a DNA methyltransferase is able to transfer the methyl group from S-adenosyl-l-methionine (AdoMet) to a cysteine residue in its catalytic center. This reaction is irreversible and relatively slow. The yield of auto-methylation is increased by addition of Dnmt3L, which functions as a stimulator of Dnmt3a and enhances its AdoMet binding. Auto-methylation was observed in binary Dnmt3a AdoMet complexes. In the presence of CpG containing dsDNA, which is the natural substrate for Dnmt3a, the transfer of the methyl group from AdoMet to the flipped target base was preferred and auto-methylation was not detected. Therefore, this reaction might constitute a regulatory mechanism which could inactivate unused DNA methyltransferases in the cell, or it could simply be an aberrant side reaction caused by the high methyl group transfer potential of AdoMet. ENZYMES: Dnmt3a is a DNA-(cytosine C5)-methyltransferase, EC 2.1.1.37. STRUCTURED DIGITAL ABSTRACT: ? Dnmt3a methylates Dnmt3a by methyltransferase assay (View interaction) ? Dnmt3a and DNMT3L methylate Dnmt3a by methyltransferase assay (View interaction).     

Rate-limiting steps in the cytidine pathway for the synthesis of phosphatidylcholine and phosphatidylethanolamine.

An analysis of the available data on the cytidine pathway for the synthesis of phosphatidylcholine and phosphatidylethanolamine, by the logic derived from the theoretical principles of metabolic regulation, shows that the first two reactions catalysed by choline (ethanolamine) kinase and phosphocholine (phosphoethanolamine) cytidylyltransferase are rate-limiting, whereas the phosphocholine (phosphoethanolamine) transferase step is near equilibrium in rat liver.     

In vivo evidence for the specificity of Plasmodium falciparum phosphoethanolamine methyltransferase and its coupling to the Kennedy pathway

Unlike humans and yeast, Plasmodium falciparum, the agent of the most severe form of human malaria, utilizes host serine as a precursor for the synthesis of phosphatidylcholine via a plant-like pathway involving phosphoethanolamine methylation. The monopartite phosphoethanolamine methyltransferase, Pfpmt, plays an important role in the biosynthetic pathway of this major phospholipid by providing the precursor phosphocholine via a three-step S-adenosyl-L-methionine-dependent methylation of phosphoethanolamine. In vitro studies showed that Pfpmt has strong specificity for phosphoethanolamine. However, the in vivo substrate (phosphoethanolamine or phosphatidylethanolamine) is not yet known. We used yeast as a surrogate system to express Pfpmt and provide genetic and biochemical evidence demonstrating the specificity of Pfpmt for phosphoethanolamine in vivo. Wild-type yeast cells, which inherently lack phosphoethanolamine methylation, acquire this activity as a result of expression of Pfpmt. The Pfpmt restores the ability of a yeast mutant pem1Deltapem2Delta lacking the phosphatidylethanolamine methyltransferase genes to grow in the absence of choline. Lipid analysis of the Pfpmt-complemented pem1Deltapem2Delta strain demonstrates the synthesis of phosphatidylcholine but not the intermediates of phosphatidylethanolamine transmethylation. Complementation of the pem1Deltapem2Delta mutant relies on specific methylation of phosphoethanolamine but not phosphatidylethanolamine. Interestingly, a mutation in the yeast choline-phosphate cytidylyltransferase gene abrogates the complementation by Pfpmt thus demonstrating that Pfpmt activity is directly coupled to the Kennedy pathway for the de novo synthesis of phosphatidylcholine.     

Automated identification of putative methyltransferases from genomic open reading frames

We have analyzed existing methodologies and created novel methodologies for the automatic assignment of S-adenosylmethionine (AdoMet)-dependent methyltransferase functionality to genomic open reading frames based on predicted protein sequences. A large class of the AdoMet-dependent methyltransferases shares a common binding motif for the AdoMet cofactor in the form of a seven-strand twisted beta-sheet; this structural similarity is mirrored in a degenerate sequence similarity that we refer to as methyltransferase signature motifs. These motifs are the basis of our assignments. We find that simple pattern matching based on the motif sequence is of limited utility and that a new method of "sensitized matrices for scoring methyltransferases" (SM2) produced with modified versions of the MEME and MAST tools gives greatly improved results for the Saccharomyces cerevisiae yeast genome. From our analysis, we conclude that this class of methyltransferases makes up approximately 0.6-1.6% of the genes in the yeast, human, mouse, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, and Escherichia coli genomes. We provide lists of unidentified genes that we consider to have a high probability of being methyltransferases for future biochemical analyses.