Adenosylmethionine Synthetase Research Articles

Knockdown of TPI in human dermal microvascular endothelial cells and its impact on angiogenesis in vitro.

Angiogenic behaviour has been shown as highly versatile among Endothelial cells (ECs) causing problems of in vitro assays of angiogenesis considering their reproducibility. It is indispensable to investigate influencing factors of the angiogenic potency of ECs. The present study aimed to analyse the impact of knocking down triosephosphate isomerase (TPI) on in vitro angiogenesis and simultaneously on vimentin (VIM) and adenosylmethionine synthetase isoform type 2 (MAT2A) expression. Furthermore, native expression profiles of TPI, VIM and MAT2A in the course of angiogenesis in vitro were examined. Two batches of human dermal microvascular ECs were cultivated over 50 days and stimulated to undergo angiogenesis. A shRNA-mediated knockdown of TPI was performed. During cultivation, time-dependant morphological changes were detected and applied for EC-staging as prerequisite for quantifying in vitro angiogenesis. Additionally, mRNA and protein levels of all proteins were monitored. Opposed to native cells, knockdown cells were not able to enter late stages of angiogenesis and primarily displayed a downregulation of VIM and an uprise in MAT2A expression. Native cells increased their TPI expression and decreased their VIM expression during the course of angiogenesis in vitro. For MAT2A, highest expression was observed to be in the beginning and at the end of angiogenesis. Knocking down TPI provoked expressional changes in VIM and MAT2A and a deceleration of in vitro angiogenesis, indicating that TPI represents an angiogenic protein. Native expression profiles lead to the assumption of VIM being predominantly relevant in beginning stages, MAT2A in beginning and late stages and TPI during the whole course of angiogenesis in vitro.

Expression of vimentin, TPI and MAT2A in human dermal microvascular endothelial cells during angiogenesis in vitro.

IntroductionIn vitro assays of angiogenesis face immense problems considering their reproducibility based on the inhomogeneous characters of endothelial cells (ECs). It is necessary to detect influencing factors, which affect the angiogenic potency of ECs.ObjectiveThis study aimed to analyse expression profiles of vimentin (VIM), triosephosphate isomerase (TPI) and adenosylmethionine synthetase isoform type–2 (MAT2A) during the whole angiogenic cascade in vitro. Furthermore, the impact of knocking down vimentin (VIM) on angiogenesis in vitro was evaluated, while monitoring TPI and MAT2A expression.MethodsA long–term cultivation and angiogenic stimulation of human dermal microvascular ECs was performed. Cells were characterized via VEGFR–1 and VEGFR–2 expression and a shRNA–mediated knockdown of VIM was performed. The process of angiogenesis in vitro was quantified via morphological staging and mRNA–and protein–levels of all proteins were analysed.ResultsWhile native cells ran through the angiogenic cascade chronologically, knockdown cells only entered beginning stages of angiogenesis and died eventually. Cell cultures showing a higher VEGFR–1 expression survived exclusively and displayed an upregulation of MAT2A and TPI expression. Native cells highly expressed VIM in early stages, MAT2A mainly in the beginning and TPI during the course of angiogenesis in vitro.ConclusionVIM knockdown led to a deceleration of angiogenesis in vitro and knockdown cells displayed expressional changes in TPI and MAT2A. Cell populations with a higher number of stalk cells emerged as being more stable against manipulations and native expression profiles provided an indication of VIM and MAT2A being relevant predominantly in beginning stages and TPI during the whole angiogenic cascade in vitro.

Biochemical characterization of a thermostable adenosylmethionine synthetase from the archaeon Pyrococcus furiosus with high catalytic power.

Adenosylmethionine synthetase plays a key role in the biogenesis of the sulfonium compound S-adenosylmethionine, the principal widely used methyl donor in the biological methylations. We report here, for the first time, the characterization of adenosylmethionine synthetase from the hyperthermophilic archaeon Pyrococcus furiosus (PfMAT). The gene PF1866 encoding PfMAT was cloned and expressed, and the recombinant protein was purified to homogeneity. PfMAT shares 51, 63, and 82% sequence identity with the homologous enzymes from Sulfolobus solfataricus, Methanococcus jannaschii, and Thermococcus kodakarensis, respectively. PfMAT is a homodimer of 90 kDa highly thermophilic with an optimum temperature of 90 °C and is characterized by remarkable thermodynamic stability (Tm, 99 °C), kinetic stability, and resistance to guanidine hydrochloride-induced unfolding. The latter process is reversible as demonstrated by the analysis of the refolding process by activity assays and fluorescence measurements. Limited proteolysis experiments indicated that the proteolytic cleavage site is localized at Lys148 and that the C-terminal peptide is necessary for the integrity of the active site. PfMAT shows kinetic features that make it the most efficient catalyst for S-adenosylmethionine synthesis among the characterized MAT from Bacteria and Archaea. Molecular and structural characterization of PfMAT could be useful to improve MAT enzyme engineering for biotechnological applications.

Cloning and Expression of Sugarcane S -adenosylmethionine Synthetase Gene ScSAM

利用RT-PCR和RACE技术从甘蔗品种新台糖22号（ROC 22）中克隆获得SAM基因的cDNA序列，命名为ScSAM，GenBank登录号为KC172558。用生物信息学方法预测分析其序列，cDNA全长1466 bp，含有1个1191 bp的完整开放阅读框（ORF），编码396个氨基酸，与高粱和玉米等植物的SAM蛋白有很高的相似性。系统进化树分析显示，甘蔗ScSAM与高粱的SAM蛋白亲缘关系较近。Real-time PCR分析表明ScSAM为组成型表达，在根中的表达量最高，是叶中表达量的3.6倍。其在黑穗病菌胁迫和低温（4℃）、聚乙二醇（PEG）、NaCl非生物胁迫下均被诱导表达，但表达模式不同；在H2O2胁迫下其表达被抑制。推测其可能参与甘蔗抗黑穗病过程，且在甘蔗抗寒、抗旱、抗盐和抗氧化等胁迫过程中也起某种作用。

Rapid and selective enzymatic assay for l-methionine based on a pyrophosphate detection system

An enzymatic assay for l-methionine was developed by coupling adenosylmethionine synthetase (AdoMetS) to a pyrophosphate (PPi) detection system, which was constructed using pyruvate, phosphate dikinase. To expand the use of this assay, the PPi detection system was embodied as three different forms, which allowed PPi to be measured by UV, visible, and fluorescent light detectors. The assay system was robust and could tolerate the addition of inorganic phosphate and ATP to the assay mixtures. l-Methionine could be accurately determined by coupling the PPi detection system and AdoMetS. This AdoMetS coupling assay was highly selective to l-methionine and exhibited no significant activity to other proteinaceous amino acids, ammonia, or urea, unlike conventional enzymatic assays for l-methionine. Spike and recovery tests showed that the AdoMetS assay could accurately and reproducibly determine increases in l-methionine in human plasma samples without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. The high selectivity and robustness of the AdoMetS assay provide rapid and high-throughput analysis of l-methionine in various kinds of analytes.

Proteome level changes in the root of Brassica alboglabra induced by alachlor herbicide

Chinese kale (Brassica alboglabra) is a famous and extensively grown vegetable in Southeast Asia. Despite its nutritional values, pesticides are heavily applied to it. In this study, changes in protein expression due to alachlor treatment on B. alboglabra were investigated by using the 2-dimensional PAGE. Differential protein expressions were determined by using Image Master Software with volume (%) ≥2 fold as significant. Ten spots of interest have been identified by LC/MS/MS showing significant increases in differential protein expression between B. alboglabra roots treated with alachlor as compared to the untreated group which include HSC-cognate binding proteins, adenosylmethionine synthetase and beta-tubulin involved in defence mechanism in plants. Little is known about the function of other proteins identified which include knox-like proteins and hypothetical protein. Further investigations on plant proteomics would provide more information on the effects of different types of pesticides.

Cloning and expression of S-Adenosyl Methionine (SAMe) Synthetase gene in recombinant E. coli strain for large scale production of SAMe

Adenosyl Methionine (SAMe) Synthetase is an enzyme which catalyses the synthesis of S-Adenosyl Methionine using methionine and ATP. It is also known as AdoMet which is well known methyl donor, which modifies DNA, RNA, histones and other proteins, dictating replicational, transcriptional and translational fidelity, mismatch repair, chromatin modeling, epigenetic modifications and imprinting. The objective of the present work is to clone the SAMe Synthetase gene in recombinant E. coli strain in order to express, characterize and purify it for further synthesis of SAMe in a large scale fermentation. Expression was induced by 1 mM IPTG and expressed protein was characterized by SDS-PAGE. The recombinant E. coli cells were used for the production of SAMe through batch and fed batch fermentation operations. The produced SAMe was purified through paper chromatography in order to use it in our future studies.

Coproporphyrin Excretion and Low Thiol Levels Caused by Point Mutation in the Rhodobacter sphaeroides S -Adenosylmethionine Synthetase Gene

A spontaneous mutant of Rhodobacter sphaeroides f. sp. denitrificans IL-106 was found to excrete a large amount of a red compound identified as coproporphyrin III, an intermediate in bacteriochlorophyll and heme synthesis. The mutant, named PORF, is able to grow under phototrophic conditions but has low levels of intracellular cysteine and glutathione and overexpresses the cysteine synthase CysK. The expression of molybdoenzymes such as dimethyl sulfoxide (DMSO) and nitrate reductases is also affected under certain growth conditions. Excretion of coproporphyrin and overexpression of CysK are not directly related but were both found to be consequences of a diminished synthesis of the key metabolite S-adenosylmethionine (SAM). The wild-type phenotype is restored when the gene metK encoding SAM synthetase is supplied in trans. The metK gene in the mutant strain has a mutation leading to a single amino acid change (H145Y) in the encoded protein. This point mutation is responsible for a 70% decrease in intracellular SAM content which probably affects the activities of numerous SAM-dependent enzymes such as coproporphyrinogen oxidase (HemN); uroporphyrinogen III methyltransferase (CobA), which is involved in siroheme synthesis; and molybdenum cofactor biosynthesis protein A (MoaA). We propose a model showing that the attenuation of the activities of SAM-dependent enzymes in the mutant could be responsible for the coproporphyrin excretion, the low cysteine and glutathione contents, and the decrease in DMSO and nitrate reductase activities.

Molecular adaptation of sourdough Lactobacillus plantarum DC400 under co-cultivation with other lactobacilli

This work was aimed at investigating the molecular mechanisms of Quorum Sensing (QS) in Lactobacillus plantarum DC400 when co-cultured with other sourdough lactobacilli. The growth and survival of L. plantarum DC400 was not affected when co-cultivated with Lactobacillus sanfranciscensis DPPMA174 or Lactobacillus rossiae A7. Nevertheless, 2-DE analysis showed that the level of protein expression of L. plantarum DC400 increased under co-culture conditions. Although several proteins were commonly induced in both co-cultures, the highest induction was found in co-culture with L. rossiae A7. Overexpressed proteins, related to QS and stress response mechanisms, were identified: DnaK, GroEL, 30S ribosomal protein S1 and S6, ATP synthase subunit beta, adenosylmethionine synthetase (MetK), phosphopyruvate hydratase, phosphoglycerate kinase, elongation factor Tu, putative manganese-dependent inorganic pyrophosphatase, d-lactate dehydrogenase, triosephosphate isomerase, fructose-bisphosphate aldolase and nucleoside-diphosphate kinase. As shown by real-time PCR, expression of the luxS gene of L. plantarum DC400 was also affected during co-cultivation. According to overexpression of MetK and luxS during co-cultivation, synthesis of AI-2-like substances was also influenced by the type of microbial co-cultures. This study showed that expression of some genes/proteins, also QS-related, in L. plantarum was influenced by co-cultivation of other sourdough lactobacilli.

Co-inhibition of Plasmodium falciparum S-Adenosylmethionine Decarboxylase/Ornithine Decarboxylase Reveals Perturbation-specific Compensatory Mechanisms by Transcriptome, Proteome, and Metabolome Analyses

Polyamines are ubiquitous components of all living cells, and their depletion usually causes cytostasis, a strategy employed for treatment of West African trypanosomiasis. To evaluate polyamine depletion as an anti-malarial strategy, cytostasis caused by the co-inhibition of S-adenosylmethionine decarboxylase/ornithine decarboxylase in Plasmodium falciparum was studied with a comprehensive transcriptome, proteome, and metabolome investigation. Highly synchronized cultures were sampled just before and during cytostasis, and a novel zero time point definition was used to enable interpretation of results in lieu of the developmentally regulated control of gene expression in P. falciparum. Transcriptome analysis revealed the occurrence of a generalized transcriptional arrest just prior to the growth arrest due to polyamine depletion. However, the abundance of 538 transcripts was differentially affected and included three perturbation-specific compensatory transcriptional responses as follows: the increased abundance of the transcripts for lysine decarboxylase and ornithine aminotransferase and the decreased abundance of that for S-adenosylmethionine synthetase. Moreover, the latter two compensatory mechanisms were confirmed on both protein and metabolite levels confirming their biological relevance. In contrast with previous reports, the results provide evidence that P. falciparum responds to alleviate the detrimental effects of polyamine depletion via regulation of its transcriptome and subsequently the proteome and metabolome.

Plant Defense Responses in Opium Poppy Cell Cultures Revealed by Liquid Chromatography-Tandem Mass Spectrometry Proteomics

Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids, including the narcotic analgesic morphine and the antimicrobial agent sanguinarine. In contrast to the plant, cell cultures of opium poppy do not accumulate alkaloids constitutively but produce sanguinarine in response to treatment with certain fungal-derived elicitors. The induction of sanguinarine biosynthesis provides a model platform to characterize the regulation of benzylisoquinoline alkaloid pathways and other defense responses. Proteome analysis of elicitor-treated opium poppy cell cultures by two-dimensional denaturing-polyacrylamide gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry facilitated the identification of 219 of 340 protein spots based on peptide fragment fingerprint searches of a combination of databases. Of the 219 hits, 129 were identified through pre-existing plant proteome databases, 63 were identified by matching predicted translation products in opium poppy-expressed sequence tag databases, and the remainder shared evidence from both databases. Metabolic enzymes represented the largest category of proteins and included S-adenosylmethionine synthetase, several glycolytic, and a nearly complete set of tricarboxylic acid cycle enzymes, one alkaloid, and several other secondary metabolic enzymes. The abundance of chaperones, heat shock proteins, protein degradation factors, and pathogenesis-related proteins provided a comprehensive proteomics view on the coordination of plant defense responses. Qualitative comparison of protein abundance in control and elicitor-treated cell cultures allowed the separation of induced and constitutive or suppressed proteins. DNA microarrays were used to corroborate increases in protein abundance with a corresponding induction in cognate transcript levels.

Cloning of A <I>S</I>-Adenosylmethionine Synthetase Gene from Broomcorn Millet (<I>Panicum miliaceum </I> L.) and Its Expression during Drought and Re-watering

Cloning of A <I>S</I>-Adenosylmethionine Synthetase Gene from Broomcorn Millet (<I>Panicum miliaceum </I> L.) and Its Expression during Drought and Re-watering

Random mutagenesis and recombination of sam1 gene by integrating error-prone PCR with staggered extension process

An efficient method for creating a DNA library is presented in which gene mutagenesis and recombination can be introduced by integrating error-prone PCR with a staggered extension process in one test tube. In this process, less than 15 cycles of error-prone PCR are used to introduce random mutations. After precipitated and washed with ethanol solution, the error-prone PCR product is directly used both as template and primers in the following staggered extension process to introduce DNA recombination. The method was validated by using adenosyl-methionine (AdoMet) synthetase gene, sam1, as a model. The full-length target DNA fragment was available after a single round. After being selected with a competitive inhibitor, ethionine, a mutated gene was obtained that increased AdoMet accumulation in vivo by 56%.

Identification of a Mutation in the Bacillus subtilis S -Adenosylmethionine Synthetase Gene That Results in Derepression of S-Box Gene Expression

Genes in the S-box family are regulated by binding of S-adenosylmethionine (SAM) to the 5' region of the mRNA of the regulated gene. SAM binding was previously shown to promote a rearrangement of the RNA structure that results in premature termination of transcription in vitro and repression of expression of the downstream coding sequence. The S-box RNA element therefore acts as a SAM-binding riboswitch in vitro. In an effort to identify factors other than SAM that could be involved in the S-box regulatory mechanism in vivo, we searched for trans-acting mutations in Bacillus subtilis that act to disrupt repression of S-box gene expression during growth under conditions where SAM pools are elevated. We identified a single mutant that proved to have one nucleotide substitution in the metK gene, encoding SAM synthetase. This mutation, designated metK10, resulted in a 15-fold decrease in SAM synthetase activity and a 4-fold decrease in SAM concentration in vivo. The metK10 mutation specifically affected S-box gene expression, and the increase in expression under repressing conditions was dependent on the presence of a functional transcriptional antiterminator element. The observation that the mutation identified in this search affects SAM production supports the model that the S-box RNAs directly monitor SAM in vivo, without a requirement for additional factors.

Cloning and Expression of Rat Liver S-Adenosylmethionine Synthetase

The S -adenosylmethionine synthetase (SAM synthetase) is responsible for in vivo synthesis of S -adenosylmethionine (SAM), which is a kind of biologically active molecules distributed in all body tissues and fluids and involved in a number of biochemical reactions. In this study, a cDNA containing the coding sequence for rat liver SAM synthetase was cloned into the prokaryotic expression vector pQE30 and expressed in E. coli M15. A major band corresponding to a protein of 48 kDa was detected on SDS-PAGE. The protein was distributed in both the soluble fraction and the insoluble fraction. In soluble fractions the protein was fully active.

Predicted highly expressed genes in the genomes of Streptomyces coelicolor and Streptomyces avermitilis and the implications for their metabolism

Highly expressed genes in bacteria often have a stronger codon bias than genes expressed at lower levels, due to translational selection. In this study, a comparative analysis of predicted highly expressed (PHX) genes in the Streptomyces coelicolor and Streptomyces avermitilis genomes was performed using the codon adaptation index (CAI) as a numerical estimator of gene expression level. Although it has been suggested that there is little heterogeneity in codon usage in G+C-rich bacteria, considerable heterogeneity was found among genes in these two G+C-rich Streptomyces genomes. Using ribosomal protein genes as references, approximately 10% of the genes were predicted to be PHX genes using a CAI cutoff value of greater than 0.78 and 0.75 in S. coelicolor and S. avermitilis, respectively. The PHX genes showed good agreement with the experimental data on expression levels obtained from proteomic analysis by previous workers. Among 724 and 730 PHX genes identified from S. coelicolor and S. avermitilis, 368 are orthologue genes present in both genomes, which were mostly 'housekeeping' genes involved in cell growth. In addition, 61 orthologous gene pairs with unknown functions were identified as PHX. Only one polyketide synthase gene from each Streptomyces genome was predicted as PHX. Nevertheless, several key genes responsible for producing precursors for secondary metabolites, such as crotonyl-CoA reductase and propionyl-CoA carboxylase, and genes necessary for initiation of secondary metabolism, such as adenosylmethionine synthetase, were among the PHX genes in the two Streptomyces species. The PHX genes exclusive to each genome, and what they imply regarding cellular metabolism, are also discussed.

Structural studies of inhibition of S-adenosylmethionine synthetase by slow, tight-binding intermediate and product analogues.

S-Adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase) catalyzes a two-step reaction in which tripolyphosphate (PPPi) is a tightly bound intermediate. Diimidotriphosphate (O(3)P-NH-PO(2)-NH-PO(3); PNPNP), a non-hydrolyzable analogue of PPPi, is the most potent known inhibitor of AdoMet synthetase with a K(i) of 2 nM. The structural basis for the slow, tight-binding inhibition by PNPNP has been investigated by spectroscopic methods. UV difference spectra reveal environmental alterations of aromatic protein residues upon PNPNP binding to form the enzyme.2Mg(2+).PNPNP complex, and more extensive changes upon formation of the enzyme.2Mg(2+).PNPNP.AdoMet complex. Stopped-flow kinetic studies of complex formation revealed that two slow isomerizations follow PNPNP binding in the presence of AdoMet, in contrast to the lower affinity, rapid-equilibrium binding in the absence of AdoMet. (31)P NMR spectra of enzyme complexes with PNPNP revealed electronic perturbations of each phosphorus atom by distinct upfield chemical shifts for each of the three phosphoryl groups in the enzyme.2Mg(2+).PNPNP complex, and further upfield shifts of at least 2 resonances in the complex with AdoMet. Comparison of the chemical shifts for the enzyme-bound PNPNP with the enzyme complexes containing either the product analogue O(3)P-NH-PO(3) or O(3)P-O-PO(2)-NH-PO(3) indicates that the shifts on binding are largest at the binding sites corresponding to those for the alpha and gamma phosphoryl groups of the nucleotide (-3.1 to -4.1 ppm), while the resonance at the beta phosphoryl group position shifts by -2.1 ppm. EPR spectra of Mn(2+) complexes demonstrate spin coupling between the two Mn(2+) in both enzyme.2Mn(2+).PNPNP and enzyme.2Mn(2+).PNPNP.AdoMet, indicating that the metal ions have comparable distances in both cases. The combined results indicate that formation of the highest affinity complex is associated with protein side chain rearrangements and increased electron density at the ligand phosphorus atoms, likely due to ionization of an -NH- group of the inhibitor. The energetic feasibility of ionization of a -NH- group when two Mg(2+) ions are bound to O(3)P-NH-PO(3) is supported by density functional theoretical calculations on model chelates. This mode of interaction is uniquely available to compounds with P-NH-P linkages and may be possible with other proteins in which multiple cations coordinate a polyphosphate chain.

Characterization of S‐adenosylmethionine synthetase genes and its expression is associated with ethylene synthesis in mustard (Brassica juncea)

S‐Adenosylmethionine synthetase (SAMS, EC 2.5.1.6) catalyses the conversion of methionine to S‐adenosylmethionine, which is a precursor of ethylene biosynthesis. To investigate whether regulation of ethylene synthesis is associated with transcript accumulation of SAMS, we report the isolation and characterization of one mustard genomic clone (MSAMS2) and two cDNAs (MSAMS3, and MSAMS4) that are highly homologous to plant SAMS. While both MSAMS2 and MSAMS3 encoded a single polypeptide of 393 amino acid residues, MSAMS4 encoded a protein of 390 residues. Accumulation of SAMS transcripts varied with mustard organs, in which transcripts were found to accumulate predominantly in root, flower bud, seedpod and leaf of the flowering plant but were less abundant in stem, petiole and leaf of the vegetative plant. SAMS transcripts could be upregulated in a gene‐specific manner in response to exogenous applications of 1‐aminocyclopropane‐1‐carboxylate, 2‐chloroethylphosphonic acid, AgNO3, polyamines (putrescine, spermidine and spermine) and NaCl. Most treatments were stimulatory to MSAMS2 and MSAMS3 transcript accumulation but salt had no effect on MSAMS2 expression. With respect to MSAMS4, the expression was generally low in control leaves and was not affected by most treatments, but exogenous applications of putrescine and spermidine upregulated transcript accumulation. The level of ethylene produced in leaves was also increased significantly in response to various stimuli, but exogenous polyamines were inhibitory to ethylene production after 12 h of incubation. These results indicate that ethylene biosynthesis in mustard is regulated, at least in part, by SAMS at the transcript level in a gene‐specific manner.

Gene sam1 encoding adenosylmethionine synthetase: effects of its expression in the fission yeast Schizosaccharomyces pombe

Gene sam1 encoding adenosylmethionine synthetase: effects of its expression in the fission yeast Schizosaccharomyces pombe

Gene sam1 encoding adenosylmethionine synthetase: effects of its expression in the fission yeast Schizosaccharomyces pombe.

By screening gene libraries of Schizosaccharomyces pombe with a DNA fragment encoding part of the Saccharomyces cerevisiae S-adenosylmethionine synthetase (SAMS), we isolated the fission yeast sam1 gene. Its sequence exhibits good homology to SAMSs of other organisms and reveals the motifs characteristic for SAMSs. SAMS activity and sam1 mRNA levels decrease when cells enter stationary phase. In haploid strains, gene sam1 is essential for growth; if weakly expressed, cells mate and sporulate at a reduced rate. Strains overexpressing sam1 exhibit methionine-sensitive growth. This methionine-induced growth inhibition is partially relieved by adenine. We assume that methionine reduces the level of one or several adenine nucleotides by a SAMS-mediated mechanism. Intracellular SAM levels increase drastically by exogenously added methionine. This increase predicts that mutants exhibiting methionine revertible phenotypes can be indicative for mutations in proteins exhibiting SAM-dependent functions. In agreement with this prediction, we show that mutant pmt2-5 has this phenotype and that gene pmt2 encodes a potential SAM-dependent enzyme.