The S-methylmethionine cycle in angiosperms: ubiquity, antiquity and activity

Angiosperms synthesize S-methylmethionine (SMM) from methionine (Met) and S-adenosylmethionine (AdoMet) in a unique reaction catalyzed by Met S-methyltransferase (MMT). SMM serves as methyl donor for Met synthesis from homocysteine, catalyzed by homocysteine S-methyltransferase (HMT). MMT and HMT together have been proposed to constitute a futile SMM cycle that stops the free Met pool from being depleted by an overshoot in AdoMet synthesis. Arabidopsis and maize have one MMT gene, and at least three HMT genes that belong to two anciently diverged classes and encode enzymes with distinct properties and expression patterns. SMM, and presumably its cycle, must therefore have originated before dicot and monocot lineages separated. Arabidopsis leaves, roots and developing seeds all express MMT and HMTs, and can metabolize [35S]Met to [35S]SMM and vice versa. The SMM cycle therefore operates throughout the plant. This appears to be a general feature of angiosperms, as digital gene expression profiles show that MMT and HMT are co-expressed in leaves, roots and reproductive tissues of maize and other species. An in silico model of the SMM cycle in mature Arabidopsis leaves was developed from radiotracer kinetic measurements and pool size data. This model indicates that the SMM cycle consumes half the AdoMet produced, and suggests that the cycle serves to stop accumulation of AdoMet, rather than to prevent depletion of free Met. Because plants lack the negative feedback loops that regulate AdoMet pool size in other eukaryotes, the SMM cycle may be the main mechanism whereby plants achieve short-term control of AdoMet level.     

Methionine metabolism in apple tissue in relation to ethylene biosynthesis

A comparison of the rate of ethylene production by apple fruit to the methionine content of the tissue suggests that the sulfur of methionine has to be recycled during its continuous synthesis of ethylene. The metabolism of the sulfur of methionine in apple tissue in relation to ethylene biosynthesis was investigated. The results showed that in the conversion of methionine to ethylene the CH₃S-group of methionine is first incorporated as a unit into S-methylcysteine. By demethylation, S-methylcysteine is metabolized to cysteine. Cysteine then donates its sulfur to form methionine, presumably through cystathionine and homocysteine. This view is consistent with the observation that cysteine, homoserine and homocysteine were all converted to methionine, in an order of efficiency from least to greatest. For the conversion to ethylene, methionine was the most efficient precursor, followed by homocysteine and homoserine. Based on these results, a methionine-sulfur cycle in relation to ethylene biosynthesis is presented.     

Cloning and expression of selenocysteine methyltransferase cDNA from <Emphasis Type="Italic">Camellia sinensis</Emphasis>

Selenocysteine methyltransferase (SMT), specifically methylates selenocysteine (SeCys) to produce the nonprotein amino acid Se-methyl selenocysteine (SeMSC) and played key role of removing selenium toxic effect at higher levels to the plant. Here we report the cloning of a cDNA encoding selenocysteine methyltransferase from Camellia sinensis (CsSMT) and expression of CsSMT in Escherichia coli. CsSMT isolated by RT-PCR and RACE-PCR reaction. CsSMT is a 1,401 bp cDNA with an open reading frame predicted to encode a 351 amino acid, 40.5 kDa protein; The predicted amino acid sequences of CsSMT shows 74% identity with A. bisulcatus selenocysteine methyltransferase (AbSMT) and 69% identity with Broccoli (Brassica oleracea var. italica) selenocysteine methyltransferase (BoSMT), and shares 53, 73 and 65% identity, respectively, with Arabidopsis thaliana homocysteine S-methyltransferase AtHMT1, AtHMT2, and AtHMT3, and 65% to Zea mays homocysteine S-methyltransferase (ZmHMT2). Analyses of CsSMT showed that it lacks obvious chloroplast or mitochondrial targeting sequences and contains a consensus sequence of GGCC for a possible zinc-binding motif near the C-terminal and a conserved Cys residue upstream of the zinc-binding motif as other related methyltransferases. Expression of CsSMT correlated with the presence of SMT enzyme activity in cell extracts, and bacteria containing recombinant CsSMT plasmid showed much high tolerance to selenate and selenite.     

Cloning and characterization of a cDNA encoding beta-amyrin synthase from petroleum plant Euphorbia tirucalli L

Euphorbia tirucalli L., known as the petroleum plant, produces a large amount of triterpenes, such as beta-amyrin. Degenerate RT-PCR based on the sequences conserved among known beta-amyrin synthases led to cloning of a putative triterpene synthase cDNA, EtAS, from leaves of E. tirucalli. The deduced amino acid sequence of the EtAS cDNA showed the highest identity of 82% to the Panax ginseng beta-amyrin synthase. Heterologous expression of the EtAS ORF in the methylotrophic yeast, Pichia pastoris, resulted in production of beta-amyrin, revealing that the EtAS cDNA codes for a beta-amyrin synthase. This is the first report of a gene involved in the triterpene synthetic pathway from Euphorbiaceae plants.     

Characterization and functional expression of cDNAs encoding methionine-sensitive and -insensitive homocysteine S-methyltransferases from Arabidopsis

Plants synthesize S-methylmethionine (SMM) from S-adenosylmethionine (AdoMet), and methionine (Met) by a unique reaction and, like other organisms, use SMM as a methyl donor for Met synthesis from homocysteine (Hcy). These reactions comprise the SMM cycle. Two Arabidopsis cDNAs specifying enzymes that mediate the SMM --> Met reaction (SMM:Hcy S-methyltransferase, HMT) were identified by homology and authenticated by complementing an Escherichia coli yagD mutant and by detecting HMT activity in complemented cells. Gel blot analyses indicate that these enzymes, AtHMT-1 and -2, are encoded by single copy genes. The deduced polypeptides are similar in size (36 kDa), share a zinc-binding motif, lack obvious targeting sequences, and are 55% identical to each other. The recombinant enzymes exist as monomers. AtHMT-1 and -2 both utilize l-SMM or (S,S)-AdoMet as a methyl donor in vitro and have higher affinities for SMM. Both enzymes also use either methyl donor in vivo because both restore the ability to utilize AdoMet or SMM to a yeast HMT mutant. However, AtHMT-1 is strongly inhibited by Met, whereas AtHMT-2 is not, a difference that could be crucial to the control of flux through the HMT reaction and the SMM cycle. Plant HMT is known to transfer the pro-R methyl group of SMM. This enabled us to use recombinant AtHMT-1 to establish that the other enzyme of the SMM cycle, AdoMet:Met S-methyltransferase, introduces the pro-S methyl group. These opposing stereoselectivities suggest a way to measure in vivo flux through the SMM cycle.     

Organoselenides from Nicotiana tabacum genetically modified to accumulate selenium

Nicotiana tabacum L. (tobacco) plants were transformed to overexpress a selenocysteine methyltransferase gene from the selenium hyperaccumulator Astragalus bisulcatus (Hook.) A. Gray (two-grooved milkvetch), and an ATP-sulfurylase gene from Brassica oleracea L. var. italica (broccoli). Solvent extraction of leaves harvested from plants treated with selenate revealed five selenium-containing compounds, of which four were identified by chemical synthesis as 2-(methylseleno)acetaldehyde, 2,2-bis(methylseleno)acetaldehyde, 4-(methylseleno)-(2E)-nonenal, and 4-(methylseleno)-(2E,6Z)-nonadienal. These four compounds have not previously been reported in nature.     

Cations modulate the substrate specificity of bifunctional class I O-methyltransferase from Ammi majus

Caffeoyl-coenzyme A O-methyltransferase cDNA was cloned from dark-grown Ammi majus L. (Apiaceae) cells treated with a crude fungal elicitor and the open reading frame was expressed in Escherichia coli. The translated polypeptide of 27.1-kDa shared significant identity to other members of this highly conserved class of proteins and was 98.8% identical to the corresponding O-methyltransferase from parsley. For biochemical characterization, the recombinant enzyme could be purified to apparent homogeneity by metal-affinity chromatography, although the recombinant enzyme did not contain any affinity tag. Based on sequence analysis and substrate specificity, the enzyme classifies as a cation-dependent O-methyltransferase with pronounced preference for caffeoyl coenzyme A, when assayed in the presence of Mg2+-ions. Surprisingly, however, the substrate specificity changed dramatically, when Mg2+ was replaced by Mn2+ or Co2+ in the assays. This effect could point to yet unknown functions and substrate specificities in situ and suggests promiscuous roles for the lignin specific cluster of plant O-methyltransferases.     

A family of S-methylmethionine-dependent thiol/selenol methyltransferases. Role in selenium tolerance and evolutionary relation

Several plant species can tolerate high concentrations of selenium in the environment, and they accumulate organoselenium compounds. One of these compounds is Se-methylselenocysteine, synthesized by a number of species from the genus Astragalus (Fabaceae), like A. bisulcatus. An enzyme has been previously isolated from this organism that catalyzes methyl transfer from S-adenosylmethionine to selenocysteine. To elucidate the role of the enzyme in selenium tolerance, the cDNA coding for selenocysteine methyltransferase from A. bisulcatus was cloned and sequenced. Data base searches revealed the existence of several apparent homologs of hitherto unassigned function. The gene for one of them, yagD from Escherichia coli, was cloned, and the protein was overproduced and purified. A functional analysis showed that the YagD protein catalyzes methylation of homocysteine, selenohomocysteine, and selenocysteine with S-adenosylmethionine and S-methylmethionine as methyl group donors. S-Methylmethionine was now shown to be also the physiological methyl group donor for the A. bisulcatus selenocysteine methyltransferase. A model system was set up in E. coli which demonstrated that expression of the plant and, although to a much lesser degree, of the bacterial methyltransferase gene increases selenium tolerance and strongly reduces unspecific selenium incorporation into proteins, provided that S-methylmethionine is present in the medium. It is postulated that the selenocysteine methyltransferase under selective pressure developed from an S-methylmethionine-dependent thiol/selenol methyltransferase.     

Expression of cytochrome c oxidase subunit 1 gene in the brain at an early stage in the termination of pupal diapause in the sweet potato hornworm, Agrius convolvuli

Suppression-subtractive hybridization was used to isolate cDNAs specifically expressed in the brain at the termination of pupal diapause in Agriusconvolvuli. One of the isolated clones shows similarity to the cytochrome c oxidase subunit 1 (COX1) gene. The full-length cDNA was obtained from brain mRNA by rapid amplification of cDNA ends (RACE). The insert is 1.65 kb in length and has an open reading frame of 1.46 kb which encodes a putative protein of 486 amino acid residues. RT-PCR reveals that the mRNA increases dramatically at an early stage of diapause termination. Activity of cytochrome c oxidase in the brain also increases at the same time. The up-regulation of this gene suggests that expression of the COX1 gene and ATP synthesis are initiated in the brain in association with diapause termination.     

Crystal structure of rat liver betaine homocysteine s-methyltransferase reveals new oligomerization features and conformational changes upon substrate binding

Betaine homocysteine S-methyltransferase (BHMT) is one of the two enzymes known to methylate homocysteine to generate methionine in the liver. It presents a Zn(2+) atom linked to three essential Cys residues. The crystal structure of rat liver BHMT has been solved at 2.5A resolution, using crystals with P2(1) symmetry and 45% solvent content in the cell. The asymmetric unit contains the whole functional tetramer showing point symmetry 222. The overall fold of the subunit consists mostly of a (alpha/beta)(8) barrel, as for human BHMT. From the end of the barrel, the polypeptide chain extends away and makes many interactions with a different subunit, forming tight dimers. The most remarkable structural feature of rat liver BHMT is the presence of a helix including residues 381-407, at the C terminus of the chain, which bind together the dimers AB to CD. A strong ion-pair and more than 60 hydrophobic interactions keep this helix stacked to the segment 316-349 from the opposite subunit. Moreover, the crystal structure of free rat liver BHMT clearly shows that Tyr160 is the fourth ligand coordinated to Zn, which is replaced by Hcy upon binding. Two residues essential for substrate recognition, Phe76 and Tyr77, are provided by a conformational change in a partially disordered loop (L2). The crucial role of these residues is highlighted by site-directed mutagenesis.     

Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B′ methyltransferase family in Coffea arabica

Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl-¹⁴C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or with any of the typical substrates of B′-MTs. It was concluded that CTgSs have strict substrate specificity. The K_m values of CTgS1 and CTgS2 were 121 and 184 μM with nicotinic acid as a substrate, and 68 and 120 μM with S-adenosyl-l-methionine as a substrate, respectively.     

Increased phloem transport of S-methylmethionine positively affects sulfur and nitrogen metabolism and seed development in pea plants

Seeds of grain legumes are important energy and food sources for humans and animals. However, the yield and quality of legume seeds are limited by the amount of sulfur (S) partitioned to the seeds. The amino acid S-methylmethionine (SMM), a methionine derivative, has been proposed to be an important long-distance transport form of reduced S, and we analyzed whether SMM phloem loading and source-sink translocation are important for the metabolism and growth of pea (Pisum sativum) plants. Transgenic plants were produced in which the expression of a yeast SMM transporter, S-Methylmethionine Permease1 (MMP1, YLL061W), was targeted to the phloem and seeds. Phloem exudate analysis showed that concentrations of SMM are elevated in MMP1 plants, suggesting increased phloem loading. Furthermore, expression studies of genes involved in S transport and metabolism in source organs, as well as xylem sap analyses, support that S uptake and assimilation are positively affected in MMP1 roots. Concomitantly, nitrogen (N) assimilation in root and leaf and xylem amino acid profiles were changed, resulting in increased phloem loading of amino acids. When investigating the effects of increased S and N phloem transport on seed metabolism, we found that protein levels were improved in MMP1 seeds. In addition, changes in SMM phloem loading affected plant growth and seed number, leading to an overall increase in seed S, N, and protein content in MMP1 plants. Together, these results suggest that phloem loading and source-sink partitioning of SMM are important for plant S and N metabolism and transport as well as seed set.     

Cloning of the novel putative apoptosis-related gene of Spirometra erinacei (Order Pseudophyllidae)

We postulated that apolysis was processed in accordance with apoptotic changes occurring in a cestode, Spirometra erinacei (Pseudophyllidea). We cloned the novel putative apoptosis-associated gene from S. erinacei via screening of a S. erinacei cDNA library with a ced-3 gene (activator of apoptosis) probe from Caenorhabditis elegans. We identified a 261-bp cDNA sequence, which encodes for an 86-amino acid protein. The cloned gene expression was observed in the neck and gravid proglottids via Northern blotting, using cloned cDNA inserts as probes, but the clone was not expressed in any of other tissues. We suggest that this gene may be involved in the apolysis of S. erinacei during normal tissue development and differentiation in cestode parasites.     

Characterization of selenocysteine methyltransferases from Astragalus species with contrasting selenium accumulation capacity

A group of selenium (Se)‐hyperaccumulating species belonging to the genus Astragalus are known for their capacity to accumulate up to 0.6% of their foliar dry weight as Se, with most of this Se being in the form of Se‐methylselenocysteine (MeSeCys). Here, we report the isolation and molecular characterization of the gene that encodes a putative selenocysteine methyltransferase (SMT) enzyme from the non‐accumulator Astragalus drummondii and biochemically compare it with an authentic SMT enzyme from the Se‐hyperaccumulator Astragalus bisulcatus, a related species that lives within the same native habitat. The non‐accumulator enzyme (AdSMT) shows a high degree of homology with the accumulator enzyme (AbSMT) but lacks the selenocysteine methyltransferase activity in vitro, explaining why little or no detectable levels of MeSeCys accumulation are observed in the non‐accumulator plant. The insertion of mutations on the coding region of the non‐accumulator AdSMT enzyme to better resemble enzymes that originate from Se accumulator species results in increased selenocysteine methyltransferase activity, but these mutations were not sufficient to fully gain the activity observed in the AbSMT accumulator enzyme. We demonstrate that SMT is localized predominantly within the chloroplast in Astragalus, the principal site of Se assimilation in plants. By using a site‐directed mutagenesis approach, we show that an Ala to Thr amino acid mutation at the predicted active site of AbSMT results in a new enzymatic capacity to methylate homocysteine. The mutated AbSMT enzyme exhibited a sixfold higher capacity to methylate selenocysteine, thereby establishing the evolutionary relationship of SMT and homocysteine methyltransferase enzymes in plants.