(Received for publication, March 17, 1995; and in revised form, July 25, 1995)
From the
The plant enzyme S-adenosylmethionine:methionine S-methyltransferase (EC 2.1.1.12, MMT) catalyzes the synthesis
of S-methylmethionine. MMT was purified 620-fold to apparent
homogeneity from leaves of Wollastonia biflora. The four-step
purification included fractionation with polyethylene glycol, affinity
chromatography on adenosine-agarose, anion exchange chromatography, and
gel filtration. Protein yield was about 180 µg/kg of leaves.
Estimates of molecular mass from sodium dodecyl sulfate-polyacrylamide
gel electrophoresis and native gel filtration chromatography were,
respectively, 115 and 450 kDa, suggesting a tetramer of 115-kDa
subunits. The 115-kDa subunit was photoaffinity labeled by S-adenosyl[H]methionine. Antibodies
raised against W. biflora MMT recognized a 115-kDa polypeptide
in partially purified MMT preparations from leaves of lettuce, cabbage,
clover, and maize.
The pH optimum of W. biflora MMT was 7.2. Kinetic analysis of substrate interaction and product inhibition patterns indicated an Ordered Bi Bi mechanism, with S-adenosylmethionine the first reactant to bind and S-adenosylhomocysteine the last product to be released. The enzyme catalyzed methylation of selenomethionine and ethionine, but not of S-methylcysteine, homocysteine, cysteine, or peptidylmethionine. Tests with other substrate analogs indicated that a free carboxyl group was required for enzyme activity, and that a free amino group was not.
The tertiary sulfonium compound S-methyl-L-methionine (SMM), ()also termed
vitamin U, occurs in a wide variety of plants at levels ranging from
about 0.01 to 6 µmol/g dry
weight(1, 2, 3, 4) . SMM is
synthesized by the action of AdoMet:Met S-methyltransferase
(MMT), which catalyzes the following
reaction(2) .
This activity has been detected in many phylogenetically diverse plants(2, 3) .
We recently demonstrated that SMM is the first intermediate in the biosynthesis of DMSP from Met in the salt-tolerant plant Wollastonia biflora(5) . DMSP is a compound with strong osmoprotectant properties that may play a role in adaptation to salt and drought stress(6) . DMSP is known to accumulate in many marine algae (7) and in a small number of flowering plants; in addition to W. biflora (family Asteraceae), these include species of Spartina(8, 9) and Saccharum(10) (family Poaceae). As for other osmoprotectant compounds(11) , genetic engineering of DMSP biosynthesis has been proposed as a strategy to enhance the stress resistance of crop plants(12) .
In plants that do not accumulate DMSP, SMM may have two other metabolic fates: reaction with Hcy to give two molecules of Met, and hydrolysis to Hse and dimethyl sulfide(1, 2) . The former reaction, catalyzed by SMM:Hcy S-methyltransferase (EC 2.1.1.10), could allow SMM to act as an efficient storage form for Met, and there is evidence for this in senescing flower tissue(13) . Mudd and Datko (3) have further proposed that tandem operation of MMT and SMM:Hcy S-methyltransferase, which they referred to as the SMM cycle, could sustain the pool of free Met in the event of an overshoot in the conversion of Met to AdoMet.
Despite its wide distribution among plants, MMT has not previously been purified to homogeneity(2, 14) . As might be expected from the high rate of SMM production required for DMSP accumulation(5) , W. biflora leaves proved to be rich in MMT. We report here the purification and characterization of the enzyme from this source. We also show that an MMT polypeptide similar in size and antigenically related to that of W. biflora is present in various plants that produce SMM but do not accumulate DMSP.
where A and B are the substrates, K and K
are the respective
limiting Michaelis constants, V is the maximum velocity, and K
K
is an interaction term.
Product inhibition data were fitted to the equations for noncompetitive (), competitive (), or uncompetitive inhibition (),
where S is the varied substrate (A or B), I is the inhibitory product, K` and V` are the apparent Michaelis constant and maximum
velocity in the presence of the product at each concentration of the
nonvaried substrate, and K
and K
are
the slope and intercept inhibition constants, respectively. The
nomenclature is that of Cleland(19) .
Figure 1: Purification of MMT from W. biflora leaves. The purification involved three column chromatographic steps: A, adenosine-agarose; B, Mono Q; and C, Superdex 200. The dotted lines are A at 280 nm, and the solid circles represent the MMT activity expressed relative to the most active fraction (100) in each chromatographic step. Activities of the most active fractions (pkat/fraction) were: A, 135; B, 490; C, 110. Estimation of native molecular mass of MMT by native gel filtration chromatography is shown in the inset. The column was calibrated with the following standard proteins: 1, thyroglobulin (670 kDa); 2, apoferritin (443 kDa); 3, alcohol dehydrogenase (150 kDa); 4, bovine serum albumin (66 kDa).
Protein fractions from the various purification steps were analyzed by SDS-PAGE (Fig. 2A). The efficiency of the affinity step is evident from comparison of lane 4 (non-retained fraction) with lane 5 (retained fraction). The latter included a major polypeptide of molecular mass 115 ± 5 kDa, which was the only one detected in the final enzyme preparation by Coomassie Blue staining (lane 7) or silver staining (not shown). Taken with the native molecular mass of 450 kDa estimated from native gel filtration chromatography, the value of 115 kDa for the denatured enzyme suggests that MMT is a tetramer of identical 115-kDa subunits.
Figure 2:
SDS-PAGE analysis of protein at various
stages of purification (A) and photoaffinity labeling of MMT
from a Mono Q fraction (B). Panel A, fractions from
each purification step were separated by SDS-PAGE and stained with
Coomassie Brilliant Blue. Lane 1, molecular mass markers
(kDa); lane 2, crude extract (80 µg); lane 3,
9-15% PEG precipitate (95 µg); lane 4, fraction not
retained by adenosine-agarose (95 µg); lane 5,
adenosine-agarose (0.7 µg); lane 6, Mono Q (0.2 µg); lane 7, Superdex 200 (0.7 µg). Panel B,
photoaffinity labeling of MMT with
[methyl-H]AdoMet. Lane 1 shows
molecular mass markers (kDa). The MMT fraction from Mono Q was
irradiated with ultraviolet light in the presence of
[methyl-
H]AdoMet with (lanes 2 and 4) or without (lanes 3 and 5) 200
µM AdoHcy. Samples were analyzed by SDS-PAGE, followed by
Coomassie Brilliant Blue staining (lanes 2 and 3) or
fluorography (lanes 4 and 5).
As an additional criterion of chemical homogeneity, purified MMT was subjected to hydrophobic interaction chromatography (Fig. 3). The purified enzyme gave a single protein peak (absorbance at 280 nm) that corresponded exactly with the peak of enzyme activity (not shown).
Figure 3:
Elution profile of purified MMT from an
alkyl Superose column. Purified MMT (50 µg) was applied to an alkyl
Superose HR 5/5 column (Pharmacia) equilibrated in buffer A containing
1.8 M (NH)
SO
, and eluted
(0.5 ml/min) with a descending gradient of
(NH
)
SO
in buffer A. The solid
line is the absorbance at 280 nm of the MMT sample; the dotted
line is that of a sample of buffer
alone.
Treatment with various thiols increased MMT activity by about 20-50% whereas the thiol reagents p-hydroxymercuribenzoate and N-ethylmaleimide strongly inhibited activity; this inhibition was reversed by dithiothreitol (Table 2). These data suggest that MMT has at least one essential Cys residue. Consistent with this, the Ser and Cys reagent phenylmethylsulfonyl fluoride (22) also inhibited MMT; however, neither iodoacetamide nor iodoacetic acid did so (Table 2). Based on these considerations, 2-mercaptoethanol was routinely added to buffers and to purified MMT preparations.
Figure 4: Double-reciprocal plots of initial reaction velocities versus AdoMet concentration at fixed concentrations of Met (A), and the corresponding slope and intercept replots (B). The quantity of enzyme was 0.7 µg/assay. In the double-reciprocal plots the points are experimental values; the lines in both panels were fitted to the points by linear regression.
Double-reciprocal plots of product inhibition data
obtained with nonsaturating concentrations of nonvaried substrate
showed that AdoHcy was a competitive inhibitor with respect to AdoMet
and noncompetitive with respect to Met, and that SMM was noncompetitive
with respect to both substrates (data not shown). At a saturating
concentration of Met (1 mM), SMM acted as an uncompetitive
inhibitor with respect to AdoMet (data not shown). These patterns rule
out a Steady State Ordered Theorell-Chance or a Rapid Equilibrium
Random mechanism but are consistent with an Ordered Bi Bi mechanism in
which AdoMet is the first substrate to bind MMT and AdoHcy the last
product released(19, 25) . Fitting the product
inhibition data to , 3, or 4, as appropriate, gave the K and K
values shown in Table 3. From these values, K
for AdoHcy and
SMM were estimated (25) as 13 ± 1 µM and
226 ± 17 µM, respectively.
Figure 5: Distribution of MMT in diverse flowering plants. MMT was partially purified from leaves as described under ``Experimental Procedures''. Approximately equal amounts of MMT activity (A) were loaded on gels, separated by SDS-PAGE, and either stained with Coomassie Brilliant Blue (B) or subjected to immunoblot analysis (C), using antibodies against MMT from W. biflora genotype H. Lane 1, W. biflora genotype H; lane 2, W. biflora genotype B; lane 3, lettuce; lane 4, red cabbage; lane 5, white clover; lane 6, maize; lane 7, prestained molecular mass markers (kDa).
This report presents the first protocol for purifying MMT to electrophoretic homogeneity. The key step, affinity chromatography on adenosine-agarose, was effective for MMT from all plants tested. With minor modification, the protocol should therefore be widely applicable.
The enzyme purified from W. biflora behaved as a homotetramer of 115-kDa subunits. Such a structure is unusual for methyltransferases, which are typically monomers or dimers with subunits of 20-45 kDa (e.g.(28, 29, 30) ). However, as an immunologically related 115-kDa polypeptide was found in MMT preparations from four other plants, the W. biflora MMT would appear to be representative. Although they lack the capacity to convert SMM to DMSP, these other plants had extractable MMT levels up to 60% of that in W. biflora. The evolution of DMSP synthesis in W. biflora may thus have involved little change in the amount of MMT, even though DMSP production would be expected to raise the net demand for SMM more than 10-fold(5) .
In addition to identifying features essential for activity as an MMT substrate, the studies with Met analogs clarified two aspects of plant metabolism. First, as previously inferred but not demonstrated(31) , the activity of MMT toward selenomethionine can account for the occurrence of Se-methylselenomethionine in plants exposed to selenium salts. Second, MMT is clearly distinct from the enzyme that catalyzes post-translational methylation of Met residues (32) because the latter has a native molecular mass of only 28 kDa; additionally, MMT did not attack Met residues in small peptides.
The properties of MMT
in general resembled those of other methyltransferases. The specific
activity of the purified enzyme (about 2.5 nkat/mg protein) is about
10-fold higher than those reported for thiol
methyltransferases(33, 34) , but falls well within the
range (about 1-10 nkat/mg) typical of small molecule
methyltransferases in general (e.g. Refs. 29, 30, 35, and 36).
Activation by thiols and inactivation by thiol reagents, implying one
or more essential Cys residues, is also a common feature among
methyltransferases(28, 37) , as is affinity labeling
by
[methyl-H]AdoMet(17, 35) .
An Ordered Bi Bi kinetic mechanism with AdoMet and AdoHcy as the
leading reaction partners would likewise be
typical(28, 38, 39) .
In W. biflora, MMT is the first enzyme in the biosynthetic pathway from Met to DMSP(5) , so its in vivo regulation is of particular significance. Some inferences about this can be drawn from our kinetic data if reasonable assumptions are made about physiological levels of the MMT substrates and products, and if the reaction catalyzed by MMT is taken to be essentially irreversible, as has been shown(2) . With respect to substrates, reports for various plants suggest concentrations of Met and AdoMet in metabolic compartments of around 100 µM(40, 41, 42) . Using these values, Fig. 6shows the extent of inhibition expected with various intracellular concentrations of AdoHcy or SMM. Although this analysis is speculative and treats each product in isolation, it suggests two points. First, if we accept the generalization (24, 43) that AdoHcy levels are normally between 25% and 100% of AdoMet levels, then it follows that MMT is likely to be inhibited in vivo by AdoHcy, and to respond sensitively to changes in AdoHcy concentration. Second, since in vivo SMM levels in W. biflora(5) are almost certainly at least 300 µM (and could be severalfold higher if SMM is located mainly in cytoplasmic compartments), then MMT is also likely to be inhibited by SMM. However, as the inhibition curve has a shallow slope, even large accumulations of SMM would be expected to produce modest inhibition. Consistent with this prediction, when W. biflora leaf disks were supplied with 5 mM external SMM, it was readily absorbed but did not depress SMM synthesis by more than about 50%(5) . As MMT was unaffected by a physiological concentration of DMSP (50 mM), it seems unlikely that the DMSP pathway is regulated by feedback inhibition at the MMT step.
Figure 6:
Inhibition of MMT by AdoHcy or SMM in the
presence of physiological concentrations (100 µM) of
AdoMet and Met. The curves were calculated (25) from (for SMM) or (for AdoHcy), using the
inhibition constants given in Table 3and the values for V, K, and K
given in the text.
Finally, our data have two implications for the proposal (12) to genetically engineer the biosynthesis of DMSP in crop plants. First, the high levels of MMT found in various plants that lack DMSP suggests that in the simplest case it would not be necessary to increase MMT expression as part of the engineering process. This assumes, of course, that the MMT of plants that lack DMSP is localized in the same subcellular compartment(s) and has kinetic properties similar to the enzyme in DMSP accumulators. The second implication concerns the level of AdoHcy, which reflects the balance between its production in transmethylation reactions and its breakdown by AdoHcy hydrolase(24, 44) . Engineering DMSP accumulation in leaf mesophyll cells might be expected to roughly triple the rate of methyl group transfer(12) , and hence the rate of AdoHcy production. In these circumstances, the endogenous AdoHcy hydrolase activity in a non-DMSP accumulating plant might not suffice to prevent AdoHcy accumulation, making it necessary to engineer higher levels of this enzyme.