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Previous Article | Next Article 
Applied and Environmental Microbiology, May 2001, p. 2044-2050, Vol. 67, No. 5
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.5.2044-2050.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Characterization of Glycine Sarcosine
N-Methyltransferase and Sarcosine Dimethylglycine
N-Methyltransferase
Antti
Nyyssölä,1,*
Tapani
Reinikainen,2 and
Matti
Leisola1
Helsinki University of Technology, Laboratory
of Bioprocess Engineering, FIN-02015 HUT Espoo,1
and Danisco Cultor Innovation, Sokeritehtaantie 20,
FIN-02460 Kantvik,2 Finland
Received 10 October 2000/Accepted 19 February 2001
 |
ABSTRACT |
Glycine betaine is accumulated in cells living in high salt
concentrations to balance the osmotic pressure. Glycine sarcosine N-methyltransferase (GSMT) and sarcosine dimethylglycine
N-methyltransferase (SDMT) of Ectothiorhodospira
halochloris catalyze the threefold methylation of glycine to
betaine, with S-adenosylmethionine acting as the methyl
group donor. These methyltransferases were expressed in
Escherichia coli and purified, and some of their enzymatic properties were characterized. Both enzymes had high substrate specificities and pH optima near the physiological pH. No evidence of
cofactors was found. The enzymes showed Michaelis-Menten kinetics for
their substrates. The apparent Km and
Vmax values were determined for all substrates
when the other substrate was present in saturating concentrations. Both
enzymes were strongly inhibited by the reaction product
S-adenosylhomocysteine. Betaine inhibited the methylation reactions only at high concentrations.
| |
INTRODUCTION |
Bacteria in saline habitats adapt to
their environment by accumulating high intracellular concentrations of
small organic compounds in order to achieve osmotic balance. These
compounds have been termed "compatible solutes" because of their
compatibility with cellular metabolism (3). Glycine
betaine (called betaine hereafter) is among the most important
compatible solutes.
The production of betaine from simple carbon sources is typical of
halophilic and halotolerant phototrophic eubacteria but rare among
heterotrophic eubacteria (30). However, many
microorganisms are able to accumulate betaine from the growth medium or
synthesize it from choline by oxidation. Also, many plants accumulate
or synthesize betaine in response to salinity or drought
(27). In plants, betaine is synthesized from choline,
which is a product of the successive methylation reactions of phospho
or phosphatidyl derivatives of ethanolamine. Choline is oxidized to
betaine in two steps, with betaine aldehyde as the intermediate
(5, 9).
We have previously shown that betaine synthesis in the extremely
halophilic anaerobic bacterium Ectothiorhodospira
halochloris proceeds via the threefold methylation of glycine in
the N position. The reactions are catalyzed by two enzymes with partly
overlapping substrate specifities. Glycine sarcosine methyltransferase
(GSMT) catalyzes the methylation steps from glycine to sarcosine
(N-monomethylglycine) and from sarcosine to dimethylglycine.
Sarcosine dimethylglycine methyltransferase (SDMT) catalyzes the steps
from sarcosine to dimethylglycine and from dimethylglycine to betaine.
S-Adenosylmethionine (AdoMet) acts as the methyl group donor
in the reactions (24).
Glycine methyltransferases have been isolated from different mammalian
origins (26). However, these enzymes are specific for
glycine; sarcosine is not methylated further (11). To our knowledge, sarcosine methyltransferases have never been described before. In this study, we have expressed these two novel enzymes in
Escherichia coli, purified them, and characterized some of their enzymatic properties.
A possible application of GSMT and SDMT is in enhancing the stress
tolerance of plants. Betaine is a well-known protectant of cells and
enzymes against various stresses (6, 27). During the past
decade, there has been considerable interest in the genetic engineering
of drought- and salt-tolerant plants. A well-established approach has
been the introduction of choline oxidation into plants which do not
synthesize betaine (20). As we have shown with E. coli, the glycine methylation pathway also has potential in improving the stress tolerance of heterologous organisms
(35) and could therefore be considered an alternative for
choline oxidation.
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MATERIALS AND METHODS |
Chemicals.
Chemicals were purchased from Sigma-Aldrich
unless otherwise indicated.
Methyltransferase activity assays.
A modification of a
previously reported methyltransferase activity assay (4)
was used. After the methylation of glycine, sarcosine, or
dimethylglycine, the unreacted methyl group donor S-adenosyl-L-[methyl-14C]methionine
is adsorbed to charcoal, and the remaining methylation products are
determined by scintillation counting. The reaction mixture typically
contained 25 µl of substrate (1 M glycine or 100 mM sarcosine in GSMT
assays and 320 mM sarcosine or 200 mM dimethylglycine in SDMT assays),
25 µl of 0.6 M Tris-HCl (pH 7.4), 25 µl of 8 mM
S-adenosyl-L-methionine iodide salt (with 91 nCi of
S-adenosyl-L-[methyl-14C]methionine
[Amersham Pharmacia Biotech]), and 25 µl of enzyme sample. The
reaction mixture was incubated at 37°C for 25 min, the reaction was
stopped by adding 75 µl of charcoal suspension (133 g/liter in 5%
[wt/vol] trichloroacetic acid), and the mixture was incubated for 10 min at 0°C. After centrifugation for 10 min, 75 µl of the
supernatant was removed for assay in a liquid scintillation counter
(model 1410; Wallac, Turku, Finland). The enzyme activity is calculated
as micromoles of methyl groups transferred per minutes.
The effect of pH on the activity of the purified enzymes was determined
by using the following buffers: 125 mM potassium phosphate (pH 4.9 to
6.2), 125 mM triethanolamine (pH 5.8 to 8.3), and 125 mM Tris-HCl (pH
8.0 to 9.0). The pH values were measured at 37°C from the reaction mixtures.
The effects of CaCl2, MgSO4, EDTA, and
p-chloromercuribenzoate were tested by incubating the
enzymes for 15 min at room temperature with 2.7 mM metal ions, 13.3 mM
EDTA, or 1.33 mM p-chloromercuribenzoate. The enzyme
reactions were started by adding the substrate (1/4 of the final
volume), and the reaction mixtures were incubated for 25 min at 37°C.
The substrate specificity was studied at 37°C using the radiometrical
methyltransferase assay described previously (25). In this
assay, the unreacted AdoMet is precipitated with phosphotungstic acid
and adenosine. The substrates used at 25 mM were ethanolamine (Fluka),
monomethylethanolamine (Fluka), and 14 of the common L-amino acids (alanine, asparagine, aspartate, cysteine,
glutamate, glutamine, isoleucine, leucine, methionine, phenylalanine,
proline, serine, threonine, and valine). The composition of the
reaction mixture and the specific radioactivity of AdoMet used at 1 mM were the same as those given above.
Construction of clones expressing GSMT and SDMT.
The
GSMT and SDMT gene fragments were amplified by
PCR with insertion of NcoI at the 5' end and
BglII at the 3' end. The primers used were 5'-CGG ACC
ATG GAT ACG ACT ACT GAG CAG-3' and 5'-GCG CAG ATC TTC AGT
CCT CCT CCC GAT ATT CCT-3' for GSMT and 5'-GCA TGC CAT GGC
GAC GCG CTA CGA CGA TCA A-3' and 5'-GCT CAG ATC TTC ACC CTT
TGC GGA AGT AAA AGA TAC-3' for SDMT. The template of the PCRs was
the plasmid used for the sequencing of the "betaine operon" of
E. halochloris (24). The amplified PCR
fragments were purified with the QIAquick DNA purification kit (Qiagen)
and cloned into NcoI/BglII-cut pQE-60 expression
vectors (Qiagen). The resulting plasmids, pGSMT and pSDMT, were
transformed into E. coli XL-1 Blue MRF' as described
previously (8). The gene sequences of the clones showing
GSMT or SDMT activity were verified at the Institute of Biotechnology
(Helsinki, Finland) by sequencing both DNA strands of the genes.
Expression of GSMT and SDMT and preparation of cell
extracts.
Fresh Luria-Bertani broth (750 ml) supplemented with 200 mg of ampicillin/liter was inoculated with 250 ml of overnight culture and grown for 30 min at 37°C and 200 rpm.
Isopropyl-
-D-thiogalactopyranoside was added to 1 mM
final concentration, and the culture was grown for a further 5 h.
The cells were separated by centrifugation (10 min; 1,500 × g), washed once with 20 mM Tris-HCl (pH 7.5), and suspended in 22 ml of 20 mM Tris-HCl (pH 7.5) supplemented with 1 mM
phenylmethylsulfonyl fluoride and 2 mM dithiothreitol. The cells were
disrupted with a Labsonic 2000 U (B. Braun, Melsungen, Germany)
sonicator in 5-ml batches with maximum power. The cell suspension was
sonicated for 5 min in 1-min pulses with intermittent cooling. The cell
debris was removed by centrifugation at 31,000 × g at
4°C for 30 min.
Purification of GSMT.
Ammonium sulfate was added to the cell
extract to achieve 25% saturation, and the solution was incubated for
45 min on ice. The suspension was centrifuged at 31,000 × g for 20 min at 0°C, and the supernatant was applied to a Butyl
Sepharose 4 FF (Amersham Pharmacia Biotech) column (1.5 by 12 cm)
preequilibrated with 25% saturated ammonium sulfate in 20 mM Tris-HCl,
pH 7.5. The column was washed with 100 ml of buffer and eluted with a
linear gradient of 25 to 0% saturated ammonium sulfate (250 ml). The active fractions (45 ml) were pooled and concentrated by
ultrafiltration (Centriplus 30; Amicon) to 10 ml. The concentrated
sample was diluted to 100 ml and applied to a DEAE Sepharose FF
(Amersham Pharmacia Biotech) column (1 by 9 cm) preequilibrated with 20 mM Tris-HCl, pH 7.5. The column was washed with 15 ml of buffer and
eluted with a linear NaCl gradient from 0 to 1 M NaCl (150 ml). The
active fractions were pooled and concentrated by ultrafiltration (Centriplus 30 [Amicon]; Ultrafree MC 10,000 NMWL [Millipore]). In
both chromatographic purification steps, GSMT eluted in approximately the middle of the gradient. The purified enzyme was supplemented with 2 mM dithiothreitol, divided into aliquots, and stored at 
80°C.
Purification of SDMT.
Ammonium sulfate fractionation at 40%
saturation was carried out as described above. The supernatant from
this purification step was diluted to 25% saturated ammonium sulfate
and applied to a Phenyl Sepharose column (high-sub) (Amersham Pharmacia
Biotech) (1.5 by 12 cm) preequilibrated with 25% saturated ammonium
sulfate in 20 mM Tris-HCl, pH 7.5. The column was washed with 100 ml of buffer and eluted with a linear gradient of 25 to 0% saturated ammonium sulfate (250 ml). The active fractions were pooled, purified further with DEAE, concentrated, and stored as for GSMT. In both chromatographic purification steps, SDMT eluted in approximately the
middle of the gradient.
HPLC analysis of the reaction products.
The reaction
products of the radiometrical assays were analyzed by high-performance
liquid chromatography (HPLC) using an Aminex HPX-87C column (Bio-Rad)
at 80°C with deionized water (0.6 ml/min) as the eluent. The specific
radioactivity of AdoMet in the reaction mixtures was from 2 to 13 times
higher than in the normal assays. The injection volumes were from 40 to
75 µl. In order to detect the methylated products, 140-µl fractions
were collected and analyzed by liquid scintillation counting as
described above.
Other methods.
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was carried out using 12% polyacrylamide
gels according to the standard protocol (16). Protein
concentrations were determined using Bio-Rad protein assay reagent with
bovine serum albumin as a protein standard. Initial-velocity data were
analyzed by nonlinear least-squares regression using the program
DYNAFIT (15). The molecular weights of GSMT and SDMT were
estimated by analytical gel filtration with a Superose 12-HR-30
(Amersham Pharmacia Biotech) column according to the instructions given
by the manufacturer. Metals were analyzed by inductively coupled plasma
emission spectrometry using an AtomScan 16 (Thermo Jarell Ash Corp.,
Franklin, Mass.) instrument at Danisco Cultor Innovation Center
(Kirkkonummi, Finland).
| |
RESULTS AND DISCUSSION |
Since it proved to be extremely difficult to purify sufficient
amounts of native GSMT and SDMT for characterization, they were
produced in E. coli. The expression levels of the enzymes were high. As estimated from SDS-PAGE, GSMT and SDMT represented roughly 30 to 50% of the total protein in the cell extracts. Both recombinant enzymes were purified to homogeneity by ammonium sulfate fractionation, hydrophobic interaction chromatography, and ion-exchange chromatography (Fig. 1). No impurities
could be detected with SDS-PAGE either when the enzyme was overloaded
or when less enzyme was loaded on the gel. The yields of the purified
recombinant enzymes were 29 and 19 mg/liter of culture for GSMT and
SDMT, respectively. The molecular masses estimated from the gel were 42 and 36 kDa for GSMT and SDMT, respectively. The value for GSMT differs
considerably from the value of 31 kDa calculated from the amino acid
sequence. However, the molecular mass of native GSMT previously
determined by SDS-PAGE was 38 kDa (24), which is also far
higher than the calculated value and reasonably close to the value
determined for recombinant GSMT in this study. The determined value for
SDMT does not differ significantly from the calculated value of 32 kDa.
Estimates for the molecular masses of the enzymes obtained by
analytical gel filtration were 40 kDa for GSMT and 25 kDa for SDMT. The
results suggest that both recombinant enzymes are monomers.
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FIG. 1.
SDS-PAGE of purified GSMT and SDMT. Lane 1, GSMT; lane
2, SDMT; lane 3, molecular mass marker. The proteins were purified by
ammonium sulfate fractionation, hydrophobic-interaction chromatography,
and ion-exchange chromatography as described in Materials and
Methods.
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Determination of pH optima.
The effects of pH on the
activities are shown in Fig. 2A for GSMT
and in Fig. 2B for SDMT. The maximal activities obtained in the range
of the triethanolamine buffer used were around pH 7.4 on glycine and
around 7.9 on sarcosine with GSMT and around 8.0 on sarcosine and
around 7.6 on dimethylglycine with SDMT. The pH optimum of SDMT appears
to depend on the buffer used. With Tris-HCl as the buffer, the optimum
for the sarcosine activity of SDMT was near pH 9.0, which differs from
the one obtained with the triethanolamine buffer. The assays were
carried out with 25 mM glycine, 25 mM sarcosine (GSMT), 80 mM sarcosine
(SDMT), or 25 mM dimethylglycine and 1 mM AdoMet as the substrates. The
results suggest that the enzyme system would work optimally near
physiological pH.
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FIG. 2.
Effect of pH on methyltransferase activities in 125 mM
potassium phosphate (approximately in the range from pH 4.9 to 6.2),
125 mM triethanolamine (approximately in the range from pH 5.8 to 8.3),
and 125 mM Tris-HCl (approximately in the range from pH 8.0 to 9.0).
(A) GSMT with 25 mM glycine ( ) and 25 mM sarcosine ( ) as the
substrates. (B) SDMT with 80 mM sarcosine ( ) and 25 mM
dimethylglycine ( ) as the substrates.
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Substrate specifity.
GSMT has strict specifity for glycine and
sarcosine, and SDMT has strict specificity for sarcosine and
dimethylglycine as the methyl group acceptors. Neither ethanolamine,
monomethylethanolamine, nor any of the L-amino acids
(alanine, asparagine, aspartate, cysteine, glutamate, glutamine,
isoleucine, leucine, methionine, phenylalanine, proline, serine,
threonine, or valine) tested at 25 mM were N methylated by GSMT or
SDMT. The results suggest that the enzymes do not catalyze the
formation of other betaines (glutamate betaine and proline betaine)
that have been reported to be components of the compatible solute pools
of cyanobacteria (18) and marine algae (19).
The results also indicate that choline, a well-known precursor of
betaine, is not formed from ethanolamine by these enzymes. High
specifity has been reported for the glycine
N-methyltransferases from rat (25) and rabbit
livers (11) as well.
Analysis of cofactors.
No evidence of cofactors was found.
Incubating GSMT or SDMT with 2.7 mM Ca2+ or
Mg2+ or 13.3 mM EDTA had no significant effect on any of
the activities. Similar results have also been reported for the
mammalian glycine methyltransferases (11). Furthermore, no
Mn, Co, or Zn and only insignificant traces of Ca and Mg were detected
when purified GSMT and SDMT were analyzed by inductively coupled plasma
emission spectroscopy.
In the UV-visible spectra (200 to 800 nm) of GSMT and SDMT, the only
peaks were at 280 nm and below 230 nm. Besides these peaks,
characteristic of all proteins, no other peaks indicating the presence
of cofactors could be detected.
Effect of p-chloromercuribenzoate acid on enzymatic
activities.
p-Chloromercuribenzoate acid (1.33 mM)
inhibited >95% of the GSMT activities on glycine and sarcosine but
only 23% of the SDMT activities on sarcosine and dimethylglycine.
With both enzymes, the inhibition was completely counteracted by the
addition of dithiothreitol (5.3 mM). The results suggest that the SH
groups play an important role in the enzyme reactions catalyzed by
GSMT. According to the amino acid sequence, both enzymes have two
cysteine residues (24).
Inhibition of enzymatic activities.
The reaction product
S-adenosylhomocysteine (AdoHcy) is known to be a strong
competitive inhibitor of many methyltransferases (e.g., see references
11, 29, and 34). The concentrations of AdoHcy that cause
50% inhibition are presented in Table 1. The results indicate that GSMT and SDMT are also very susceptible to
inhibition by AdoHcy and that its efficient removal is required for
betaine synthesis to continue in living cells. This can be achieved by
S-adenosylhomocysteine hydrolase-catalyzed hydrolysis of
AdoHcy to adenosine and homocysteine (31). The
inhibitory effects of dimethylglycine on GSMT and of glycine on
SDMT were also studied. These compounds were not methylated by the
enzymes under the conditions used. As shown in Table 1,
dimethylglycine, the product of the methylation of sarcosine, was a
relatively poor inhibitor of GSMT. Also, glycine inhibited the
activities of SDMT only at high concentrations. At 50 mM it had no
effect on sarcosine activity but inhibited 27% of dimethylglycine
activity. At 200 mM, it inhibited 13% of the sarcosine activity and
56% of the dimethylglycine activity. The results indicate that the affinity of glycine to SDMT is low.
In phototrophically growing E. halochloris, the
intracellular betaine concentrations vary from about 0.7 to 1.6 mol/kg
of water at a salinity range from 120 to 240 g/kg of water
(7). The intracellular K+ and Na+
concentrations (0.33 M K+ and 0.66 M Na+)
remain fairly constant at these salinities (33). The
relative activities of GSMT and SDMT when betaine and KCl (0.33 M)-NaCl (0.66 M) were present in the reaction mixtures are shown in Table 2. KCl-NaCl inhibited all methylation
steps to various degrees. Betaine is known to protect many enzymes from
the perturbing effects of salts and other stress factors
(6). However, betaine did not relieve the inhibiting
effects of KCl-NaCl on GSMT or SDMT but inhibited the reactions in high
concentrations. At 2 M, it inhibited all enzyme activities, both with
KCl-NaCl present and with it absent.
Without KCl-NaCl, 0.5 M betaine had no effect on the first methylation
step and a small activating effect on the later steps of the pathway.
The concentration of 0.5 M is higher than the reported levels of
betaine in salt-stressed plants and plant cell organelles
(27), which indicates that in the concentrations naturally
found in plants, GSMT and SDMT would not be inhibited by betaine.
Sibley and Yopp (31) have proposed that the regulation of
betaine synthesis in the halophilic cyanobacterium Aphanothece halophytica is based on the (putative) strong inhibitory effect of
AdoHcy on the methyltransferases catalyzing betaine biosynthesis in
this strain. Briefly, according to this model, betaine synthesis is
regulated by the different effects of various intracellular K+ and betaine concentrations on the hydrolytic or
synthetic activity of AdoHcy hydrolase. The results presented in Table
2 suggest that salts and betaine can also directly affect the
activities of the methyltransferases. The regulation of the methylation
pathway is presumably very complicated, and the data presented here do not allow a detailed analysis of it.
Kinetic properties.
GSMT catalyzes the reaction sequence
glycine-sarcosine-dimethylglycine, and SDMT catalyzes the sequence
sarcosine-dimethylglycine-betaine. In all the two-substrate reactions,
AdoMet acts as the methyl group donor. The apparent kinetic parameters
for both substrates of every reaction step were determined with the
other substrate present in excess. GSMT and SDMT displayed
Michaelis-Menten kinetics for their substrates. The sigmoidal rate
behavior with respect to AdoMet reported for the mammalian
glycine-methyltransferases was not detected in any of the initial
velocity patterns. The apparent Km and
Vmax values determined are shown in Table
3.
As presented in Table 3, in every methylation step, the
Vmax values determined for both substrates are
close to each other. The initial velocity data fitted reasonably well
with the hyperbolic curves with both high and low substrate
concentrations, a fact that is also indicated by the standard errors
presented in Table 3. However, we cannot completely rule out the
possibility of some minor substrate inhibition resulting from the
saturating concentrations of the cosubstrate used.
We have previously purified native GSMT from E. halochloris
and reported its specific activities on glycine and sarcosine. These
results were obtained with 25 mM glycine, sarcosine, or dimethylglycine
and 1 mM AdoMet as the substrates (24). According to the
results presented in Table 3, some of the substrates were not present
in saturating concentrations in these assays. This appears to be
especially true for the activities on glycine. Reexamining the
linearity of the product formation in the enzymatic reactions revealed
that our previous results were not obtained in the strictly linear
range. With the activity on glycine, there is also the possibility that
the strong inhibition by AdoHcy distorted the results, because the
AdoMet concentration was close to its Km value.
Nevertheless, the specific activities of recombinant GSMT and SDMT were
determined under the same conditions used earlier in order to compare
the activities with our previous results. The specific activities of
purified GSMT were 0.16 µmol min
1 mg1 on
glycine and 0.075 µmol min1 mg1 on
sarcosine, and those of purified SDMT were 0.68 µmol
min1 mg1 on sarcosine and 3.4 µmol
min1 mg1 on dimethylglycine.
The specific activities of recombinant GSMT were 3.3 and 2.5 times
lower than those reported for native GSMT, with glycine and sarcosine,
respectively, as the substrates (24). The deviations of
the product formation rates from linearity do not explain the differences between the specific activities. The clones were verified by sequencing, and therefore it is also highly unlikely that the lower
activity would be the result of a mutation during the PCR amplification. One possibility is that the low specific activity of
recombinant GSMT is a result of incorrect folding of the enzyme due to
differences between the intracellular conditions for E. coli
and those for E. halochloris. However, taking into account the error margins, the ratios of the activities on different substrates correspond reasonably well to our previous results for native GSMT and
also for His6-tagged GSMT and SDMT (24).
Our experiences with the expression of GSMT and SDMT in
Saccharomyces cerevisiae have been similar to those with
E. coli. In S. cerevisiae, the activities of GSMT
were extremely low compared to those of SDMT, although high levels of
both enzymes (identified by Western blotting) were synthesized (A. Nyyssölä, unpublished results). It would therefore seem
likely that the heterologous expression of GSMT in plants would also
present difficulties.
Since both enzymes catalyze two successive steps, it was necessary to
discover whether the second methylation step would distort the results
of the initial velocity determinations of the first step. Using the
apparent Km and Vmax
values for the sarcosine step of GSMT, it was estimated that the
methylation of the newly formed sarcosine had only a negligible effect
on the initial velocity determinations with glycine as the substrate.
This was also confirmed by HPLC analyses (see Materials and Methods) of
reactions with 4 mM AdoMet and 250 mM glycine, 4 mM AdoMet and 9 mM
glycine, and 1 mM AdoMet and 25 mM glycine as substrates. In these
reactions, the amounts of dimethylglycine formed were only 3 to 7% of
the total products (sarcosine plus dimethylglycine). The reaction times
were such that the final product concentrations were considerably higher than those in any of the initial velocity determinations.
The situation is different with the SDMT activity on sarcosine. As
shown in the HPLC analysis of the reaction mixture (Fig. 3A), the newly formed dimethylglycine is
readily methylated to betaine with 4 mM sarcosine and 4 mM AdoMet as
the substrates. Consequently, reliable values for the apparent
Km and Vmax for the
sarcosine activity with a fixed AdoMet concentration cannot be obtained
by the methods used in this study.
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FIG. 3.
HPLC analysis of radioactive methylation products with
SDMT. The radioactive products were determined from fractions collected
from the eluent as described in Materials and Methods. (A) Reaction
with 4 mM sarcosine as the substrate. (B) Reaction with 80 mM sarcosine
as the substrate. The methyl group donor
S-adenosyl-L-[methyl-14C]methionine
was used at 4 mM in both reactions. The retention times are shown by
triangles on the x axis (D, dimethylglycine; B, betaine).
The retention times in panels A and B are not comparable.
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However, with 4 mM AdoMet and an excess of sarcosine (80 mM) as
substrates, the amount of betaine formed was negligible (Fig. 3B).
Also, with 0.11 and 1 mM AdoMet at this sarcosine concentration, only
barely detectable amounts of betaine were formed (data not shown). The
results indicate that when saturating concentrations of sarcosine are
used, the reaction catalyzed by SDMT stops at the dimethylglycine
stage. It would seem plausible that under these conditions sarcosine
competitively inhibits the further methylation of the newly formed
dimethylglycine to betaine. Because of this phenomenon, we were able to
determine the apparent kinetic parameters for the AdoMet activity with
a saturating concentration of sarcosine as the cosubstrate (Table 3).
Although the initial velocities of SDMT are unreliable when determined
with less than a saturating concentration of sarcosine, the apparent
kinetic parameters with sarcosine as the variable substrate were
estimated. With the shortest possible reaction times, the apparent
Km and Vmax determined
were 6.1 mM and 1.3 µmol min1 mg1,
respectively (Table 3). The Vmax is close to the
value determined with the sarcosine concentration fixed and the AdoMet
concentration variable, most likely because of the inhibition of the
second step by high concentrations of sarcosine. From the estimate of the apparent Km for sarcosine, nothing more can
be concluded than that it appears to be of the same order of magnitude
as the Km for dimethylglycine of SDMT.
The apparent Km value of 18 mM determined for
glycine was significantly greater than the ones determined for the
other substrates and also greater than those reported for the mammalian
glycine methyltransferases (ranging from 0.13 mM for rat to 11 mM for pig) (25, 26). However, the apparent
Vmax of the glycine methylation step is six
times greater than the value for the rat glycine methyltransferase (25). Also, the apparent Km for
AdoMet for the glycine step (0.42 mM) is higher than that for the other
steps and higher than the S0.5 values (the
substrate concentration at which the reaction rate is half of its
maximal value) of the mammalian glycine methyltransferases (around 0.3 mM for human, rabbit, and pig and 0.05 mM for rat) (26).
The apparent Km values for AdoMet decrease
somewhat in the successive methylation steps. This suggests that AdoMet
is more efficiently used when the reaction to betaine proceeds.
Possible applications of heterologous expression of GSMT and
SDMT.
The glycine methylation pathway has many possible
applications. Betaine is widely used as an additive in the feed
industry. Thus, transgenic plants producing high concentrations of
betaine would have a better nutritional value and could therefore be
used directly in feed without the supplementation of betaine. As
already mentioned, the stress-relieving potential of betaine is not
limited to plants. Many industrial microbes suffer from environmental stress under processing conditions. For example, the high substrate concentrations used in fermentation media can cause osmotic stress. The
glycine methylation pathway could therefore also be used in improving
the stress tolerance of commercially important microbes in agriculture
and industry (T. Reinikainen, A. Nyyssölä, and J. Kerovuo,
March 2000, U.S. patent application 09/137, 434).
The most interesting possibility, however, is the application of GSMT
and SDMT in the genetic engineering of stress-tolerant plants. Drought,
salinity, and low temperatures are among the most important
environmental factors limiting plant productivity (2). The
stress-relieving effects of betaine on plants and its stabilizing
effects on plant macromolecules have been widely reported (13,
14, 21, 27, 36). Many important crops, such as rice, potato,
tomato, and tobacco, do not synthesize betaine, and therefore,
introducing betaine synthesis into these plants has been a
well-established target for metabolic engineering (20). So
far, the strategy has been the utilization of the choline oxidation pathway. Enzymes catalyzing the oxidation of choline to betaine have
been introduced into many plants (1, 10, 12, 17, 22, 28).
However, the levels of betaine in the transgenic plants have been
significantly lower than those in plants that naturally accumulate it,
although in some cases improved stress tolerance has been reported.
It is believed that the reason for the low betaine contents of the
transgenic plants is the limited supply of choline (22). Presumably, the reactions of choline metabolism in
non-betaine-producing plants form a rigid metabolic network, which
makes it difficult to direct choline to betaine synthesis
(23). In higher plants, the ethanolamine for choline
synthesis is produced from glycine via serine (32).
Bypassing the glycine-choline pathway by the introduction of GSMT and
SDMT would make it possible to directly engineer all three methylation
reactions of betaine synthesis and conceivably to avoid the
difficulties associated with the choline oxidation pathway.
It remains to be seen how efficient the glycine methylation pathway is
against choline oxidation. We hope that the information on the basic
characteristics of GSMT and SDMT presented in this study will
facilitate future work on introducing these methyltransferases into
plants. In addition to E. halochloris, there are many other halophilic bacteria synthesizing betaine de novo (30), and
it is reasonable to assume that the majority of them use the glycine methylation pathway as well. Consequently, there are most likely abundant alternatives to the GSMT and SDMT of E. halochloris
in nature.
| |
ACKNOWLEDGMENTS |
We are grateful to Markku Saloheimo and VTT Biotechnology for
letting us use their scintillation counter. We also thank Xiaoyan Wu,
Niklas von Weymarn, and especially Martti Marjamaa for their kind help
with the analyses.
This work was supported by the Research Foundation of Helsinki
University of Technology and by Danisco Cultor.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Helsinki
University of Technology, Laboratory of Bioprocess Engineering, P.O.
Box 6100, FIN-02015, HUT, Finland. Phone: 358-9-4512544. Fax:
358-9-462373. E-mail: antti.nyyssola{at}hut.fi.
| |
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Applied and Environmental Microbiology, May 2001, p. 2044-2050, Vol. 67, No. 5
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.5.2044-2050.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
