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J. Biol. Chem., Vol. 275, Issue 21, 15962-15968, May 26, 2000

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Characterization and Functional Expression of cDNAs Encoding Methionine-sensitive and -insensitive Homocysteine S-Methyltransferases from Arabidopsis^*

Philippe Ranocha, Fabienne Bourgis, Michael J. Ziemak, David Rhodes^§, Douglas A. Gage^¶, and Andrew D. Hanson

From the  Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611, the ^§ Department of Horticulture, Purdue University, West Lafayette, Indiana 47907, and the ^¶ Biochemistry Department, Michigan State University, East Lansing, Michigan 48824

Received for publication, February 10, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Unlike other organisms, plants synthesize L-S-methylmethionine (SMM)¹ from Met and S-adenosylmethionine (AdoMet) in a reaction mediated by AdoMet:Met S-methyltransferase (MMT, EC 2.1.1.12) (1-3). SMM can then serve as a methyl donor for the synthesis of Met from homocysteine (Hcy) catalyzed by Hcy S-methyltransferase (HMT, EC 2.1.1.10). The tandem action of MMT and HMT, plus that of S-adenosylhomocysteine (AdoHcy) hydrolase, constitutes the SMM cycle (Fig. 1). Although MMT and the SMM cycle are unique to plants, HMT occurs in bacteria, yeast, and mammals, enabling them to catabolize SMM of plant origin and providing an alternative to the methionine synthase reaction as a means to methylate Hcy (4-7).

In wheat and other plants, SMM is synthesized in leaves and transported via the phloem to developing seeds where it can be used to methylate Hcy (8). SMM is also synthesized by morning glory flower buds and then used to methylate Hcy during blooming (9). The halves of the SMM cycle can thus sometimes be separated in space or time. However, both halves may also operate concurrently in the same tissue, and in these cases the cycle has been hypothesized to remove excess AdoMet (3). Testing this hypothetical homeostatic role, which is analogous to that of the cyclic methylation/demethylation of Gly in mammalian liver (10), requires determination of flux through the SMM cycle in defined tissues in vivo. Methods to do this are lacking.

The first enzyme of the SMM cycle, MMT, has been purified from Wollastonia biflora and barley, and characterized (2, 11). MMT cDNAs have been isolated from W. biflora, Arabidopsis, and maize, and the two latter plants have been shown to have one MMT gene (8). Much less is known about plant HMTs, and none has been cloned from plants or other eukaryotes. HMT was partially purified from jack beans and germinating peas (12, 13) and shown to be stereoselective for one of the two methyl groups of SMM (the pro-R methyl) (14). The preparations obtained used either SMM or AdoMet as methyl donor; it was not established whether both activities reside on the same protein. These data appear to indicate that plants can bypass SMM by recycling AdoMet methyl groups directly to Met (Fig. 1, dotted arrows). However, the AdoMet substrates used in these experiments most probably contained significant levels of the nonphysiological R,S diastereomer (15), and it has been suggested that this, not the physiological S,S form, is the substrate for HMTs (7). The form of AdoMet that plant HMTs utilize is therefore unclear. Neither jack bean nor pea HMT was strongly inhibited by Met (25% inhibition by 10 mM Met; Refs. 12 and 13), which contrasts with the Met sensitivity of the yeast enzyme (12).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   The S-methylmethionine cycle and related reactions in higher plants. The two core reactions of the cycle are shown by bold arrows. Dotted arrows indicate a possible shorter cycle in which AdoMet donates a methyl group to Hcy. Note that this scheme has three different Hcy  Met reactions; their Hcy substrates are, for simplicity, shown separately but may derive from the same pool in vivo. THF, tetrahydrofolate; CH3-THF, 5-methyltetrahydrofolate.

Recently, the Escherichia coli YagD protein was shown to be an HMT, and a similar enzyme, selenocysteine Se-methyltransferase (SeCysMT), was characterized and cloned from the selenium-accumulating plant Astragalus bisulcatus (7, 16, 17). These enzymes share significant primary sequence homology (7) and have a GGCC motif near the C terminus. The cysteine residues in this motif have been implicated in zinc binding in two other enzymes that catalyze methyl transfers to Hcy, E. coli B₁₂-dependent Met synthase and mammalian betaine-Hcy methyltransferase (18, 19). The enzyme-bound zinc is required to activate the thiol group of Hcy for nucleophilic attack (18).

In this work, we identified two Arabidopsis homologs of YagD and confirmed that they encode HMTs. The recombinant enzymes were partially characterized, with emphasis on clarifying their substrate specificity and sensitivity to Met. We also surveyed the genomic complexity of HMT genes in Arabidopsis and used the known stereoselectivity of HMT to establish that of the other enzyme of the SMM cycle, MMT. The results of the stereospecificity study suggest a novel approach to determining flux through the SMM cycle in vivo.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals and Reagents-- L-[³⁵S]Met (>800 Ci mmol¹) and L-[methyl-¹⁴C]AdoMet (58 mCi mmol¹; in 10 mM H₂SO₄, ethanol, 9:1) were from NEN Life Science Products; L-[1-¹⁴C]Met (55 mCi mmol¹) was from American Radiolabeled Chemicals (St. Louis, MO), and D-[1-¹⁴C]Met (56 mCi mmol¹) was from Moravek Biochemicals (Brea, CA). L-[³⁵S]SMM (10 µCi nmol¹) and D- and L-[1-¹⁴C]SMM were synthesized from radiolabeled Met and purified as described previously (20); radiochemical purities were >99%. L-[U-¹³C₅]Met (97-98% ¹³C abundance) was from Cambridge Isotope Laboratories (Andover, MA). (6R,6S)5-Methyltetrahydrofolate was obtained from Schirks Laboratories and 3-dimethylsulfoniopropionate (DMSP) from Research Plus (Bayonne, NJ); other biochemicals were from Sigma. L-SMM iodide was either converted to the chloride using Dowex-1 (Cl) (for enzyme assays) or freed of traces of Met and converted to the free base using Dowex-1 (OH) and then neutralized with HCl (for growth media). D- and L-Hcy were freshly prepared from the thiolactone hydrochlorides (21). DL-Selenocysteine was prepared immediately before use by reduction of DLselenocystine with NaBH₄ (17); the product was verified by TLC on cellulose developed in n-butanol:acetic acid:water (6:2:2, v/v). Recombinant Arabidopsis MMT was prepared as described (8). Ion exchange resins were from Bio-Rad. Cellulose (0.1 mM) TLC plates were from Merck, and silica gel G (0.25 mm) plates were from Machery-Nagel.

Separation of AdoMet Diastereomers-- The S,S (biologically active) and R,S (inactive) diastereomers of AdoMet were separated by HPLC essentially as described by Beaudouin et al. (22). Analytical scale separations of [methyl-¹⁴C]AdoMet and unlabeled AdoMet were made on a 1 × 150 mm Reliasil C18 column using a microbore HPLC system (UMA model, Michrom Bioresources, Auburn, CA). Solvent A was water containing 0.1 M sodium acetate, 20 mM citric acid, 0.93 mM octanesulfonic acid, and 0.12 mM EDTA; solvent B was methanol, and the gradient was from 100-95% solvent A in 45 min. The elution profile was monitored at 258 nm. The [methyl-¹⁴C]AdoMet contained no detectable R,S form (1% or less) and was used without further purification. As previously reported (15), unlabeled AdoMet was found to contain 15% of the R,S isomer. In the few cases (see "Results") in which unlabeled AdoMet was included in enzyme assays, specific radioactivity calculations were based on its (S,S)-AdoMet content.

E. coli and Saccharomyces cerevisiae Strains, Plasmids, and Growth Conditions-- The E. coli strain used in complementation tests was MTD123 (yagD metE metH) (16) and the expression vector was pBluescript SK- (Stratagene). The minimal medium was M9 (24) containing 0.8% glucose, L-Met, or L-SMM (70 µM) and 1 mM isopropyl -D-thiogalactopyranoside. The S. cerevisiae strains CY61-1A (MAT his3 leu2 ura3 ade2 trp1 met6::HIS3) and CY61-1D (MATa his3 leu2 ura3 ade2 trp1 met6::HIS3 ypl273::URA3 yll062::HIS3) were obtained from Y. Surdin-Kerjan (Centre de Génétique Moléculaire, CNRS, Gif-sur-Yvette, France). The yeast expression vector was pVT102-L (25). The synthetic minimal medium for yeast and the culture conditions were as described (26) except for the inclusion of adenine (100 µM) and L-SMM or AdoMet (100 µM).

cDNA Generation, Sequencing, and Sequence Analysis-- Arabidopsis expressed sequence tags, GenBank^TM accession numbers T46013 and H37463 (encoding AtHMT-1 and -2, respectively), were obtained from the Arabidopsis Biological Resource Center (Columbus, OH). The 750-base pair insert in H37463, which is truncated at the 5'-end, was used to isolate a full-length cDNA from an Arabidopsis (ecotype Landsberg erecta) leaf library in the  Uni-Zap XR vector (Stratagene) (provided by T. L. Thomas, Texas A&M University). DNA sequencing procedures were as described (8). Sequence alignments were made using Clustal W 1.7 (27); phylogenetic analysis was carried out using the Darwin system at the ETH server. Homology searches were made using BLAST programs (28).

cDNA Expression in E. coli-- HMT coding sequences were amplified from plasmid templates by high fidelity polymerase chain reaction using recombinant Pfu DNA polymerase (Stratagene). The primers included the first or last 6-7 codons plus restriction sites for cloning into pBluescript SK- and, for the forward primers, a Shine-Delgarno sequence preceded by a stop codon in frame with the LacZ protein encoded by the vector. The AtHMT-1 primers were 5'-CGGAATTCTTGAAGGAAACAGCTATGGTTTTGGAGAAAAAATC-3' (forward) and 5'-CCCAAGCTTTCATCTTCGTTTCAAATCTC-3' (reverse); the AtHMT-2 primers were 5'-AAAACTGCAGGTGAAGGAAACAGCTATGACCGGAAACTCTTTTAAC-3' (forward) and 5'-CGGGGTACCCTAAAGAGATCTGCGGTTGAC-3' (reverse). After ligation into pBluescript, constructs were introduced into E. coli strain DH10B by electroporation. Plasmid preparations were sequenced to verify the inserts and used to transform E. coli strain MTD123 by electroporation.

cDNA Expression in Yeast-- HMT coding sequences were amplified as above using primers that included the first or last eight codons plus BamHI and PstI restriction sites for cloning into pVT102-L. pVT102-L contains the leu2-d gene for selection and the ADH1 promoter to drive gene expression (25). The AtHMT-1 primers were 5'-CGCGGATCCATGGTTTTGGAGAAAAAATCTGC-3' (forward) and 5'-AAAACTGCAGTCATCTTCGTTTCAAATCTCTGG-3' (reverse); AtHMT-2 primers were 5'-CGCGGATCCATGACCGGAAACTCTTTTAACTC-3' (forward) and 5'-AAAACTGCAGCTAAAGAGATCTGCGGTTGACGA-3' (reverse). After ligation into pVT102-L, constructs were introduced into E. coli strain DH10B by electroporation. Constructs were verified by sequencing and used to transform strain CY61-1D as described (25).

Enzyme Isolation and Molecular Mass Determination-- E. coli cultures (50 ml) were grown to an A₆₀₀ of 0.6-1 in LB medium (24) containing 100 µg ml¹ ampicillin and 1 mM isopropyl-1-thio--D-galactopyranoside. Cells were harvested by centrifugation (4000 × g, 10 min, 4 °C), washed in buffer A (100 mM Hepes-KOH, pH 7.5, 1 mM DTT, 10% glycerol), recentrifuged, frozen in liquid N₂, and stored until extraction at 80 °C. Subsequent operations were at 0-10 °C. Cells were resuspended in buffer A (5 ml/50-ml culture) and broken by sonication; the extract was cleared by centrifugation (10,000 × g, 15 min) and used for enzyme assays directly or after desalting on PD-10 columns (Amersham Pharmacia Biotech) equilibrated in buffer A. Extracts were routinely stored at 80 °C after freezing in liquid N₂; this was shown not to affect HMT activity. Yeast extracts were prepared as described previously (26) using buffer A. Native molecular masses were estimated using a Waters 626 HPLC system equipped with a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech); reference proteins were cytochrome c, carbonic anhydrase, bovine serum albumin, and -amylase. Protein was estimated by Bradford's method (29) using bovine serum albumin as standard.

Enzyme Assays-- Unless otherwise indicated, assays were made under conditions in which substrates were saturating and product formation was proportional to enzyme level and time. The assays were modifications of that described by Mudd and Datko (3). SMM:Hcy S-methyltransferase assays (final volume 50 µl) contained 20 mM Hepes-KOH buffer, pH 7.5, 2 mM DTT, 2 mM Hcy (or other methyl acceptor), 200 µM [³⁵S]SMM (15-25 nCi/assay), and enzyme extract. AdoMet:Hcy S-methyltransferase assays were similar except that the final volume was 5 µl, and the Hepes-KOH concentration was raised to 200 mM (due to the H₂SO₄ in the [methyl-¹⁴C]AdoMet preparation); except where noted, no unlabeled AdoMet was included. Reactions were incubated at 30 °C for 30 min and stopped by adding 1 ml of ice-cold 1 mM Met solution. The diluted mixtures were applied to 0.5-ml columns of Dowex-50 (NH₄⁺), which were washed with 6 ml of water; the effluent was mixed with 5 ml of scintillation fluid (Beckman Ready Gel) and counted. Assay blanks contained no methyl acceptor. The [³⁵S]Met formed in SMM:Hcy methyltransferase assays was analyzed by TLC on silica gel G in methanol:acetone:HCl (90:10:4, v/v/v) before and after oxidation to the sulfoxide by treating with 30% H₂O₂ for 1 h at 24 °C.

Diastereospecificity Experiments-- ¹³C-Labeled SMM was synthesized in a 1-ml reaction mixture containing 10 mM potassium phosphate buffer, pH 7.2, 1 mM DTT, 10% glycerol, 1 µmol of L-[U-¹³C₅]Met, 1.5 µmol of AdoMet, and 10 picokatal of recombinant Arabidopsis MMT activity. After incubation for 16 h at 30 °C, the mixture was passed through 1-ml Dowex-1 (OH) and BioRex-70 (H⁺) columns arranged in series. Met, AdoMet, and AdoHcy were retained by the Dowex-1 column; the [¹³C]SMM (yield = 0.39 µmol) was eluted from BioRex-70 with 5 ml of 1 M HCl and lyophilized. The [¹³C]SMM was then used as a substrate for HMT. The 1-ml reaction mixtures contained 10 mM potassium phosphate buffer, pH 7.5, 1 mM DTT, 10% glycerol, 0.39 µmol of [¹³C]SMM, 1.5 µmol of L-Hcy, and desalted E. coli extract containing 2 nanokatal of AtHMT-1 activity. After incubation for 4 h at 30 °C, the mixture was passed through 1-ml columns of Dowex-50 (NH₄⁺) and Dowex-50 (H⁺) arranged in series. Residual SMM was retained on the first column; the [¹³C]Met product (yield = 0.35 µmol) was eluted from the second column with 5 ml of 6 M NH₄OH and lyophilized. The [¹³C]Met was separated from peptidoglycan in the lyophilizate by extraction in 95% ethanol and dried in vacuo.

Electrospray Mass Spectrometry-- The [¹³C]Met formed by the sequential action of MMT and HMT was analyzed on a FinniganMAT LQC (Thermoquest, San Jose, CA) mass spectrometer system. The source voltage was set at 3.5 kV and capillary voltage at 30 V; the capillary temperature was 22 °C. Background source pressure was 1.5 × 10⁵ torr as read by an ion gauge. The sample flow rate was 10 µl min¹. The drying gas was N₂. The LQC was scanned to 2000 atomic mass units. Spectra were acquired for 0.5 min. Samples were dissolved in 50 µl of water; 25 µl was injected into the mass spectrometer.

DNA Gel Blot Analyses-- Arabidopsis genomic DNA was isolated from leaves as described (30). Five-µg samples of the isolated DNA were digested, separated in 0.7% agarose gels, and transferred to supported nitrocellulose membrane (Nitropure, MSI) as described (24). Blots were hybridized overnight at 58 °C in 5× SSC, 5× Denhardt's solution, 1% SDS, 1 mM EDTA, and 100 µg ml¹ sonicated salmon sperm DNA and washed at low stringency (1× SSC, 0.1% SDS, 37 °C). The probes were the full-length AtHMT-1 or -2 cDNAs and were labeled with ³²P by the random primer method. Radioactive bands were detected by autoradiography.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Genomic-based Cloning of HMT cDNAs from Arabidopsis-- BLAST searches using the amino acid sequence of E. coli YagD detected two sets of homologous Arabidopsis expressed sequence tags. Sequencing one insert from each set (GenBank^TM accession numbers T46013 and H37463) established that they represent two distinct transcripts. The T46013 insert encodes a 326-residue (36.0 kDa) polypeptide, designated AtHMT-1. The H37463 insert encodes only the C-terminal part of a polypeptide and so was used to isolate the corresponding full-length cDNA from an Arabidopsis leaf library. This cDNA specifies a 333-residue (36.4 kDa) polypeptide, designated AtHMT-2. The deduced AtHMT-1 and -2 proteins are 55% identical to each other, 50 (AtHMT-1) or 68% (AtHMT-2) identical to Astragalus SeCysMT, and 24-41% identical to YagD and two yeast proteins, Ypl273w and Yll062c, that were shown to be HMTs while our work was in progress (Fig. 2).² AtHMT-1 and -2 also share significant sequence identity (20-26%) with the N-terminal region of E. coli B₁₂-dependent Met synthase and with mammalian betaine-Hcy methyltransferase (not shown). AtHMT-1 and -2 both have a GGCC zinc-binding motif near the C terminus, as well as a third conserved cysteine sited 65 residues upstream that may also be a zinc ligand (18, 19). Both AtHMT-1 and -2 appear to lack targeting sequences (e.g. chloroplast or mitochondrial transit peptides), indicating that they are cytosolic enzymes.

View larger version (77K): [in this window] [in a new window]   Fig. 2.   Alignment of the deduced amino acid sequences of Arabidopsis HMTs with related methyltransferases from other organisms. Identical residues are shaded in black, and similar residues are gray. Dashes are gaps introduced to maximize alignment. The bar indicates the core of the zinc-binding motif reported in B12-dependent Met synthase and betaine-Hcy methyltransferase (18, 19). The asterisk marks a third conserved cysteine residue. AtHMT-1 and -2, Arabidopsis HMT-1 and -2; YagD, E. coli YagD (BAA12002); Yll062c, S. cerevisiae Yll062c (S50958); Ypl273w, S. cerevisiae Ypl273w (S65306); SecysMT, A. bisulcatus selenocysteine Se-methyltransferase (CAA10368).

Complementation of an E. coli yagD Mutant and Detection of HMT Activity-- The coding regions of AtHMT-1 and -2 were subcloned into pBluescript SK-. To express the HMTs as native proteins and not LacZ fusions, the coding sequences were preceded by a stop codon in frame with LacZ and a Shine-Delgarno sequence. These constructs were introduced into E. coli strain MTD123 (yagD metE metH), which lacks Met synthase and HMT activity, and is consequently a Met auxotroph that cannot grow on SMM (16). Both constructs enabled transformants to grow on SMM (Fig. 3A); no transformants grew on medium without SMM, indicating complementation of the yagD mutation and not the metE or metH mutations (not shown). No complementation was observed with the vector alone (Fig. 3A), and retransforming MTD123 with rescued plasmids containing the AtHMT-1 or -2 cDNAs restored the ability to grow on SMM, showing that the complementation is because of the encoded plant protein. HMT activity was readily detected in extracts of the complemented strains but not, as expected, in cells transformed with the vector alone (Fig. 3B). The specific activity of AtHMT-1 was 10-fold higher than that of AtHMT-2; this difference was observed consistently in independent experiments. To authenticate the observed activities, the [³⁵S]Met reaction products were verified by TLC (Fig. 3C).

View larger version (56K): [in this window] [in a new window]   Fig. 3.   Complementation of an E. coli yagD mutant by Arabidopsis HMT cDNAs and HMT activities in complemented strains. A, similar numbers of cells of E. coli K12 (wild-type) (1), the yagD metE metH mutant MTD123 (2), and MTD123 transformed with pBluescript SK- containing AtHMT-1 (3) or AtHMT-2 (4), or alone (5) were plated on minimal medium containing 1 mM isopropyl-1-thio--D-galactopyranoside and 70 µM Met or SMM. B, HMT (L-SMM:L-Hcy methyltransferase) activities in extracts of MTD123 transformed with pBluescript (pBS) alone or carrying AtHMT-1 or -2 cDNAs. Data are mean ± S.E. (n = 3-7). C, autoradiograph of a TLC separation of the [35S]Met reaction product before () and after (+) H2O2 treatment to convert it to Met sulfoxide (MetSO). The positions of the origin (Ori) and of authentic standards are marked.

Methyl Acceptor Specificity of AtHMT-1 and -2-- We compared the ability of AtHMT-1 and -2 to catalyze methyl transfer from L-SMM to various thiols and related compounds, using L-Hcy as the benchmark (Table I). Both enzymes utilized D-Hcy, although AtHMT-1 showed a marked preference for the L form. A similar lack of stereospecificity toward Hcy has been noted for other HMTs (7, 31). AtHMT-1 showed significant activity with L- and D-cysteine, which is noteworthy as cysteine is not a substrate for E. coli or yeast HMTs (7, 31). Neither enzyme attacked DL-selenocysteine (Table I), glutathione, coenzyme A, sulfide, or thiocyanate (not shown).

View this table: [in this window] [in a new window]   Table I Methyl acceptor specificity of AtHMT-1 and -2  Desalted extracts of E. coli strain MTD123 (yagD metE metH) expressing AtHMT-1 or -2 were assayed for activity using L-[35S]SMM (0.2 mM) as methyl donor and various acceptors at a concentration of 2 mM. Data are means of three replicates ± S.E.

Methyl Donor Specificity of AtHMT-1 and -2-- SMM occurs in plants as the L enantiomer (8) and AtHMT-1 and -2 both proved to be specific for this form: with 20 µM D- or L-[1-¹⁴C]SMM and 2 mM L-Hcy as substrates, activities with D-SMM were undetectable (<3% of those with L-SMM). To compare L-SMM and (S,S)-AdoMet as methyl donors, Michaelis constants and relative V_max values were determined for both enzymes (Table II). (S,S)-AdoMet was found to be a methyl donor for both enzymes, but the K_m values were higher than for L-SMM (67-fold for AtHMT-1 and 4.5-fold for AtHMT-2) and the V_max values were lower. The (S,S)-[methyl-¹⁴C]AdoMet used in these experiments contained no detectable (1%) R,S diastereomer and, in the assay conditions used (pH 7.5, 30 min), 0.3% (R,S)-AdoMet is expected to form by racemization (15). (R,S)-AdoMet therefore did not contribute significantly to the observed activities. Because SMM and (S,S)-AdoMet are substrates, K_m values for L-Hcy were determined with both (Table II); fairly similar values were obtained with both methyl donors and with both enzymes. To screen for other potential methyl donors, unlabeled compounds were tested for their ability to inhibit methyl transfer from [³⁵S]SMM when added to assays in 5-fold molar excess. Glycine betaine, choline, phosphocholine, DMSP, and 5-methyltetrahydrofolate had little effect on either enzyme (13% inhibition; data not shown), making it unlikely that they are significant methyl donors.

Met Sensitivity and Other Biochemical Properties of AtHMT-1 and -2-- With either L-SMM or (S,S)-AdoMet as a methyl donor, AtHMT-1 activity showed strong product inhibition by L-Met, whereas AtHMT-2 did not, being almost unaffected by L-Met concentrations in the physiological range (500 µM) (Fig. 4). AtHMT-1 and -2 both showed maximal activity at pH 7.5. Neither was stimulated by Zn²⁺ (0.1 or 1 mM). AtHMT-1 activity was modestly inhibited by 1 mM EDTA (26%); AtHMT-2 activity was not. The molecular masses of the native AtHMT-1 and -2 enzymes were estimated by size exclusion chromatography to be 36 kDa. This indicates that both enzymes exist as monomers, as do other HMTs and SecysMT (7, 17, 31).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Inhibition of homocysteine methyltransferase activities by the product L-methionine. Activities were measured using physiological concentrations of L-Hcy (20 µM) and L-SMM or (S,S)-AdoMet (100 µM) (estimated from Refs. 1, 8, and 33-36, assuming these metabolites to be confined to a cytoplasmic compartment occupying 5% of the cell). A, SMM:Hcy S-methyltransferase activities; activities in the absence of L-Met were 10.9 and 5.7 nmol min1 mg1 protein for AtHMT-1 and -2, respectively. B, (S,S)-AdoMet:Hcy S-methyltransferase activities; activities in the absence of L-Met were 0.025 and 0.020 nmol min1 mg1 protein for AtHMT-1 and -2, respectively. , AtHMT-1; , AtHMT-2.

Complementation of Yeast HMT Mutations-- Yeast cells take up SMM and AdoMet and metabolize them to Met via the action of HMT (5, 6). Disruption of the open reading frames yll062c (MHT1) and ypl273w (SAM4) (Fig. 2) has demonstrated that they specify HMTs that prefer SMM and AdoMet, respectively.² A triple disruptant (CY61-1D) lacking Met synthase as well as both HMTs is consequently a Met auxotroph that cannot use SMM or AdoMet in place of Met. To confirm that AdoMet and SMM serve as methyl donors for AtHMT-1 and -2 in vivo, each was expressed in CY61-1D and the transformants were tested for the ability to grow on SMM or AdoMet (Fig. 5) (this type of experiment cannot be carried out in E. coli because it cannot absorb AdoMet). AtHMT-1 and -2 enabled growth on either compound establishing that SMM and AdoMet are indeed substrates for both enzymes in vivo as well as in vitro. To exclude the possibility that the differing Met sensitivities shown in Fig. 4 are an artifact of expression in E. coli, SMM:Hcy S-methyltransferase activity was assayed in desalted extracts of yeast transformants expressing AtHMT-1 and -2. As with the recombinant enzymes from E. coli, AtHMT-1 was inhibited strongly by L-Met (92% at 500 µM L-Met), whereas AtHMT-2 was not. Activities without L-Met were 1.9 and 1.8 nmol min¹ mg¹ protein for AtHMT-1 and -2, respectively; these nearly equal values contrast with the 10-fold difference seen when these enzymes are expressed in E. coli (Fig. 3B). These data suggest that AtHMT-2 may be less stable (or synthesized more slowly) than AtHMT-1 in the bacterial host.

View larger version (51K): [in this window] [in a new window]   Fig. 5.   Complementation of yeast HMT mutations. Similar numbers of cells of strain CY61-1A (met6) (1), the met6 ypl273 yll062 mutant CY61-1D (2), and CY61-1D transformed with pVT102-L containing AtHMT-1 (3) or AtHMT-2 (4), or alone (5) were plated on minimal medium containing 0.1 mM Met, L-SMM, or AdoMet. The AdoMet used was not purified to remove the R,S diastereomer, because in the conditions used, this forms continuously in the medium from racemization of (S,S)-AdoMet (15).

Diastereospecificity of Methyl Transfer in the SMM Cycle-- Because HMT is known to transfer the pro-R methyl group of SMM to Hcy (14), we used recombinant AtHMT-1 to determine the diastereospecificity of MMT, the other enzyme of the SMM cycle. To do this, L-[U-¹³C₅]Met and unlabeled AdoMet were used as substrates for recombinant Arabidopsis MMT; the SMM formed in this reaction was then incubated with unlabeled L-Hcy and AtHMT-1. The resulting ¹³C-labeled Met was analyzed by electrospray MS, together with a 1:1 mixture of unlabeled Met and L-[U-¹³C₅]Met for comparison (Fig. 6). The product of the MMT/HMT reactions gave peaks of almost equal intensity at m/z 151 and 154, corresponding to [¹³C₁]Met and [¹³C₄]Met, and no appreciable signal above that expected for natural abundance ¹³C, ¹⁵N, and ³³S at m/z 155 ([¹³C₅]Met). The small peak at m/z 150 (unlabeled Met) is attributable to ¹²C in the original [¹³C₅]Met substrate (Fig. 6B). These data show that MMT introduces a methyl group into the pro-S position of SMM, i.e. that MMT and HMT have opposite stereoselectivities.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Electrospray MS analysis of [13C]Met derived from the sequential action of MMT and HMT, and of Met and [13C5]Met standards. A, 13C-Labeled SMM was synthesized from [U-13C5]Met and unlabeled AdoMet using Arabidopsis MMT, then used together with unlabeled Hcy as a substrate for AtHMT-1. The mass spectrum shown is of the Met formed in the latter reaction. B, an equimolar mixture of unlabeled Met and the [U-13C5]Met used in the experiment shown in A. The peak at m/z 154 is attributable to 12C present in the [U-13C5]Met preparation.

Genomic Complexity and Relationships of HMT Genes in Arabidopsis-- Southern blot analyses carried out at low stringency indicated that both AtHMT-1 and AtHMT-2 are encoded by single genes (Fig. 7, A and B). Consistent with this result, BLAST searches of the Arabidopsis genome (84% complete at the time of searching) revealed a chromosome III sequence specifying AtHMT-1 (AB023041, nucleotides 21893-23610) but no other closely related sequences. Molecular phylogenic analysis (Fig. 7C) of the sequences aligned in Fig. 2 suggests (a) that AtHMT-2 and Astragalus SecysMT belong on a branch distinct from AtHMT-1, and (b) that extant HMTs are derived from a single ancestral gene that existed prior to the divergence of eubacteria and eukaryotes and has undergone independent duplications in plant and yeast lineages.

View larger version (41K): [in this window] [in a new window]   Fig. 7.   Southern blot analysis of HMT Genes. Genomic DNA was isolated from Arabidopsis leaves, digested with the restriction enzymes indicated, separated on a 0.7% agarose gel (5 µg/lane), blotted, and probed sequentially with the complete AtHMT-1 cDNA (A) and AtHMT-2 cDNA (B). Washing was at low stringency. The sizes of hybridizing bands match the cDNAs with respect to the predicted restriction sites. Genomic reconstruction standards were made with AtHMT-1 and -2 cDNAs equivalent to 1 and 5 copies/haploid genome (shown on the left of each panel). Note that AtHMT-1 and -2 cDNAs cross-hybridize only very weakly as they differ at 55% of the base pairs. The positions of DNA size markers (kb, kilobase) are marked. Abbreviations are as in Fig. 2. C shows a molecular phylogenic tree of the protein sequences from Fig. 2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The identification of cDNAs encoding plant HMTs completes the set of genes required for operation of the SMM cycle, the others being MMT and AdoHcy hydrolase (3). This opens the way to comprehensive studies of the expression of these genes and to the systematic application of reverse genetics to probe the function of SMM and its cycle. Furthermore, extracts of E. coli expressing AtHMT-1 or -2 have specific activities 10²-fold higher than those of the best plant sources (12, 13) making them good material for future enzyme purification. More generally, the HMT cDNAs reported here appear to be the first identified from a eukaryote.

AtHMT-1 and -2 resemble HMTs from other organisms in overall primary structure and in being monomeric proteins. They lack obvious targeting sequences and are therefore presumably cytosolic enzymes. HMT has yet to be definitively localized in plant cells, but preliminary work with pea leaves indicates that it is cytosolic,³ as are other enzymes involved in Met metabolism, i.e. MMT, Met synthase, AdoMet synthetase, and AdoHcy hydrolase (8, 32). AtHMT-1 and -2 share with other HMTs and with SecysMT, a GGCC zinc-binding motif (18), plus a third conserved cysteine residue. This strongly suggests that they have a zinc cofactor. Neither enzyme was stimulated by zinc or severely inhibited by EDTA, but this may be because the zinc is tightly bound, as it is in betaine-Hcy methyltransferase (19).

Our results demonstrate that the physiological S,S diastereomer of AdoMet is a substrate for plant HMTs. This indicates that plants have the potential to bypass SMM by transferring methyl groups directly from AdoMet to Hcy (Fig. 1, dotted arrows), and the complementation experiments with yeast confirm that plant HMTs can mediate this reaction in a foreign host. But how much flux does this bypass actually carry in planta? Kinetic considerations indicate that it may be very little, especially in tissues where AtHMT-1 is the predominant isoform. AtHMT-1 has K_m values for SMM and AdoMet of 29 and 1950 µM, respectively and the V_max value with SMM is 2.8-fold higher. SMM levels are reported to range from about 5 to >300 nmol g¹ fresh weight in various tissues, and SMM/AdoMet ratios are reported to range from 1 to >30 (1, 8, 33-36). Some SMM may be sequestered in the vacuole; however, radiotracer kinetic studies indicate that the metabolically active (presumably cytosolic) SMM pool is a large fraction of the total (36). Assuming the cytosol to be 5% of tissue water volume (37), it follows from these data that typical cytosolic SMM concentrations are likely to be 100 µM, and AdoMet concentrations are likely to be similar or lower. In such conditions flux through the AdoMet-driven reaction would be 3% of that through the SMM-driven reaction. Simply put, a high prevailing SMM concentration can deny AdoMet access to the AtHMT-1 active site and thereby suppress futile cycling of AdoMet.

Our finding that AtHMT-1 is strongly inhibited by Met is novel, because the plant HMTs so far known are Met-insensitive (12, 13). Met sensitivity may be crucial to the control of flux through the HMT reaction and the SMM cycle. A Met-sensitive HMT could stop the cycle turning when Met levels are elevated, whereas a Met-insensitive enzyme could allow SMM  Met conversion even when Met levels are high. It is therefore noteworthy that free Met levels in developing seeds can greatly exceed those in other tissues (400 versus 10-30 nmol g¹ fresh weight) (1, 35, 38-40) and that HMTs isolated from seeds are Met-insensitive (12, 13). Moreover, DNA array data indicate that the predominant HMT expressed in developing Arabidopsis seeds is the Met-insensitive AtHMT-2.⁴ Another difference between the Arabidopsis HMTs is that AtHMT-1 attacks cysteine. This could explain the origin of S-methylcysteine in the Brassicaceae. No enzyme that catalyzes the S-methylation of cysteine has hitherto been demonstrated (1), although radiotracer data show that the reaction occurs in vivo (41).

The SMM cycle has been proposed to rectify overshoots in the conversion of free Met to AdoMet, thereby sustaining a free Met pool for protein synthesis (3). This hypothesis was based largely on data for whole Lemna plantlets (3), and it has since been found that SMM is transported between organs in the phloem (8). This raises the question of whether the SMM was produced and utilized in the same organs in the Lemna experiments and shows that accurate flux measurements are now needed to clarify the functions of the SMM cycle. Only a few such measurements have been made, and these come from unusual plants (W. biflora and Spartina alterniflora) that convert SMM to DMSP. Isotope tracer studies of SMM synthesis and metabolism in leaves of these plants showed that the methyl flux from Met to SMM was high, but there was little or none from SMM to Hcy, i.e. the SMM cycle turned slowly if at all (36, 42). The approach used to make these measurements depends on the metabolism of SMM to DMSP and so unfortunately cannot be applied to the great majority of plants that do not synthesize DMSP.

There is thus a need for methods to estimate flux through the SMM cycle in tissues of non-DMSP-accumulating plants. Our finding that the enzymes of the cycle have opposing stereoselectivities suggests a novel way to do this. For example, consider an organ that imports SMM via the phloem and ultimately uses it to produce Met that is used for protein synthesis. If the SMM cycle is not operating, then supplied SMM that has a ¹³C label in the C₄ backbone and the pro-R methyl and ²H₃ label in the pro-S methyl will give rise to only two labeled species of Met in proteins: [methyl-²H₃,¹³C₄]Met and [methyl-¹³C]Met. However, if the SMM cycle is operating, the additional species [methyl-²H₃]Met, [¹³C₄]Met, and [¹³C₅]Met will be found in proteins and will become relatively more abundant with each turn of the cycle.

	ACKNOWLEDGEMENTS

We thank Dr. Y. Surdin-Kerjan for the gift of yeast strains CY61-1A and -1D, for permission to refer to unpublished data and for helpful discussions, Dr. A. Böck for E. coli strain MTD123, and Dr. T. L. Thomas for the gift of the Arabidopsis cDNA library.

	FOOTNOTES

^* This work was supported in part by National Science Foundation Grants IBN-9816075 (to A. D. H) and IBN-9904263 (to D. A. G.), by Department of Energy Grant DE-FG02-99ER20344 (to D. R.), by an endowment from the C. V. Griffin, Sr. Foundation, and by the Florida Agricultural Experiment Station. Journal series no. R-07506.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF219222 (AtHMT-1) and AF219223 (AtHMT-2).

To whom correspondence should be addressed: Horticultural Sciences Dept., University of Florida, P. O. Box 110690, Gainesville, FL 32611. Tel.: 352-392-1928; Fax: 352-392-6479; E-mail: adha@gnv. ifas.ufl.edu.

Published, JBC Papers in Press, March 21, 2000, DOI 10.1074/jbc.M001116200

² D. Thomas, A. Becker, and Y. Surdin-Kerjan, personal communication.

³ P. Ranocha and A. D. Hanson, unpublished data.

⁴ J. B. Ohlrogge, personal communication.

	ABBREVIATIONS

The abbreviations used are: SMM, S-methylmethionine; AdoMet, S-adenosylmethionine; MMT, S-adenosylmethionine:methionine S- methyltransferase; HMT, S-methylmethionine (or S-adenosylmethionine):homocysteine S-methyltransferase; AdoHcy, S-adenosylhomocysteine; SeCysMT, selenocysteine Se-methyltransferase; DMSP, 3-dimethylsulfoniopropionate; HPLC, high performance liquid chromatography; DTT, dithiothreitol.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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