From the Research Institute and § Graduate
School of Environmental and Human Sciences, Meijo University,
Nagoya 468-8502, Japan, ¶ Shimadzu Company, Nakagyou-ku, Kyoto,
604-8511, Japan, and the
Graduate School of Agricultural
Science, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
Received for publication, October 28, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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Glycine betaine
(N,N,N-trimethylglycine) is an
important osmoprotectant and is synthesized in response to abiotic
stresses. Although almost all known biosynthetic pathways of betaine
are two-step oxidation of choline, here we isolated two
N-methyltransferase genes from a halotolerant
cyanobacterium Aphanothece halophytica. One of gene
products (ORF1) catalyzed the methylation reactions of glycine and
sarcosine with S-adenosylmethionine acting as the methyl
donor. The other one (ORF2) specifically catalyzed the methylation of
dimethylglycine to betaine. Both enzymes are active as monomers.
Betaine, a final product, did not show the feed back inhibition for the
methyltransferases even in the presence of 2 M. A reaction
product, S-adenosyl homocysteine, inhibited the methylation
reactions with relatively low affinities. The co-expressing of two
enzymes in Escherichia coli increased the betaine level and
enhanced the growth rates. Immunoblot analysis revealed that the
accumulation levels of both enzymes in A. halophytica cells increased with increasing the salinity. These results indicate that
A. halophytica cells synthesize betaine from glycine by a three-step methylation. The changes of amino acids Arg-169 to Lys or
Glu in ORF1 and Pro-171 to Gln and/or Met-172 to Arg in ORF2
significantly decreased Vmax and increased
Km for methyl acceptors (glycine, sarcosine, and
dimethylglycine) but modestly affected Km for
S-adenosylmethionine, indicating the importance of these
amino acids for the binding of methyl acceptors. Physiological and
functional properties of methyltransferases were discussed.
The most known biosynthetic pathways of betaine are the two-step
oxidation of choline. Many bacteria, plants, and animals accumulate
glycine betaine (here after betaine) under abiotic stress conditions
(1-3). In these organisms, it was shown that betaine is synthesized by
two steps, choline It was suggested that betaine might be synthesized from glycine by a
series of methylation reactions in archaebacterium
Methanohalophilus portucalensis (12) and
anaerobic phototrophic sulfur bacterium Ectothiorhodospira
halochloris (13). Betaine synthesis from simple carbon sources has
also been suggested in aerobic heterotrophic eubacterium
Actinopolyspora halophila (13) and halotolerant cyanobacterium of Aphanothece halophytica (14).
Recently, the methyltransferase genes that are involved in betaine
synthesis have been isolated from E. halochloris and
A. halophila (15). Two methyltransferase genes were involved
in E. halochloris. One of gene products catalyzed the
methylation reactions of glycine and sarcosine to sarcosine and
dimethylglycine, respectively
(EcGSMT),1 whereas the other
one catalyzed the methylations of sarcosine and dimethylglycine to
dimethylglycine and betaine, respectively (EcSDMT) (15, 16). By
contrast, one ORF was found in A. halophila of which
the N- and C-terminal parts had homologous sequences to those of EcGSMT
and EcSDMT, respectively (15). The functionality of A. halophila methyltransferase was not well shown due to the formation of cell pellet when expressed in Escherichia
coli.
Glycine N-methyltransferase (GMT) catalyzing the methylation
of glycine to sarcosine is known in mammalian cells although the
enzymes catalyzing the further methylation steps do not occur (17, 18).
The homology of amino acid sequences between mammalian GMT and EcGSMT
was low. No homologous sequences to those of EcGSMT, EcSDMT, and
A. halophila methyltransferase could be found. Therefore, it
was interesting to examine whether betaine is synthesized from glycine
by three-step methylation reactions in other organisms.
A. halophytica is an oxygen-evolving halotolerant
cyanobacterium that can grow in a wide range of salinity conditions
from 0.25 to 3.0 M NaCl concomitant with the accumulation
of betaine (14, 20). Previous studies have shown that A. halophytica has unique systems to survive under severe
environmental conditions (20-24). Ribulose-1,5-bisphosphate
carboxylase/oxygenase of A. halophytica dissociates easily
into large and small subunits when betaine was absent (20). A. halophytica DnaK has been shown to contain the longer C-terminal
segment than other DnaK/Hsp70 family members (21) and exhibit extremely
high protein folding activity at high salinity (22). It was also shown
that A. halophytica Na+/H+
antiporter has novel ion specificity (23) and could confer the
tolerance for salt stress of fresh water cyanobacterium capable of
growing in sea water (24). Here, we show that in contrast to other
oxygen-evolving photosynthetic organisms, A. halophytica synthesizes betaine from glycine by a three-step methylation. Two novel
methyltransferase genes were isolated, and their functional properties
were examined. From the mutagenesis approach, the information on the
substrate binding sites in both enzymes was obtained.
Culture Conditions--
E. coli DH5 Isolation of Methyltransferase Genes--
The genomic DNA of
A. halophytica was partially sequenced by a shotgun method
using a Shimadzu multicapillary DNA sequencing system (Shimadzu Co.,
Kyoto, Japan). Homology searches were made using the BLAST program as
previously described (23). Two methyltransferase genes, ORF1 and ORF2,
homologous to EcGSMT and EcSDMT were found and amplified by the PCR
reactions using the genomic DNA of A. halophytica as a
template. The forward primers for ORF1 and ORF2 were ORF1-F and ORF2-F,
respectively and contain the start codon ATG and the NcoI
restriction site (Table I). The reverse
primers for ORF1 and ORF2 were ORF1-R and ORF2-R, respectively, and
contain the stop codon and the BamHI restriction site. The
amplified fragments for ORF1 (798 bp) and ORF2 (834 bp) were ligated
into EcoRV restriction site of pBlueScript SK+. The
resulting plasmids, pORF1SK+ and pORF2SK+, were transferred to E. coli DH5
Expression vectors for ORF1 and ORF2 were constructed using a pET3d
plasmid (Novagen). For this, pORF1SK+ and pORF2SK+ were digested with
NcoI and BamHI and ligated into the
NcoI- and BamHI-double digested pET3d vector. The
resulting plasmids were designated as pORF1 and pORF2, respectively.
For the co-expression of ORF1 and ORF2, the plasmid pORF1 was
double-digested with BglII and BamHI. The
resulting fragment containing the full-length of ORF1 together with the
T7 promoter region was ligated into the BamHI-digested site
of pORF2. The resulting plasmid containing both ORF1 and ORF2 was
designated as pORF12.
Construction of Arg-169 Mutants in ORF1--
The R169K and R169E
mutants of ORF1 were constructed by the PCR technique using the plasmid
pORF1SK+ as template. For the R169K mutant, the C-terminal part of ORF1
was amplified by the forward primer, ORF1-R/KF, and reverse primer,
ORF1-R. The forward primer, ORF1-R/KF, corresponds to the 493-525 bp
of ORF1 and contains the introduced XbaI site and AAG (Lys)
in place of CGG (Arg). The N-terminal part of ORF1 was amplified by the
forward primer, ORF1-F and the reverse primer, ORF1-R/DKR. The reverse
primer, ORF1-R/DKR corresponds to the 476-502 bp and contains
the introduced XbaI site. The amplified fragments were
mixed, annealed, and then once again amplified by using the forward and
reverse primer sets, ORF1-F and ORF1-R, respectively. The amplified
fragment was ligated into pBluescript II SK+ and sequenced. The correct
clone was transferred to pET3d and used for the expression in BL21
cells. The R169E mutant was constructed essentially with the same
methods as those of the R169K mutant except for the change of ORF1-R/KF
with ORF1-R/DF.
Construction of Pro-171 and Met-172 Mutants in ORF2--
The
P171Q, M172R, and P171Q/M172R mutants of ORF2 were constructed by the
PCR technique using the plasmid pORF2SK+ as template. The C-terminal
part of ORF2 was amplified by the forward primer, ORF2-PM/QRF, and
reverse primer, ORF2-R. The forward primer, ORF2-PM/QRF, corresponds to
the 504-533 bp of ORF2 and contains the mixed nucleotides C(C/A)AA(T/G)G, which encode Pro- or Asn-171 and Met- or Arg-172. The
N-terminal part of ORF2 was amplified by the forward primer, ORF2-F,
and the reverse primer, ORF2-PM/QRR. The reverse primer, ORF2-PM/QRR
corresponds to the nucleotides 492-521 bp of ORF2. The amplified
fragments were mixed, annealed, and then once again amplified by using
the forward and reverse primer sets, ORF2-F, and ORF2-R, respectively.
The amplified fragment was ligated into pBluescript II SK+ and
sequenced. The correct clones were transferred to pET3d and used for
the expression in BL21 cells.
Expression and Purification of Methyltransferase Genes in E. coli--
The plasmids, pORF1, pORF2, and pORF12, were transferred to
E. coli BL21 (DE3). Cells expressing ORF1 and ORF2 were
grown in LB medium containing 50 µg/ml ampicillin until
A at 620 nm reached 0.6-0.8. Then, 0.5 mM isopropyl-
The ORF1 protein was precipitated by addition of ammonium sulfate (25%
saturation). The precipitated fraction was dissolved in Buffer A and
dialyzed against the same buffer A. The dialyzed soluble fraction was
applied to a TSK BioAssist Q column (4.6 mm × 5.0 cm). The ORF1
protein was eluted with a gradient of 0-1000 mM NaCl.
Active fractions were pooled, dialyzed against buffer A, and applied to
the same column. After three times of column chromatography, the
purified ORF1 protein was obtained. For the purification of ORF2
protein, the cell extract was fractionated with ammonium sulfate
(50-75% saturation). The precipitated fraction was dissolved in
Buffer A, dialyzed against the same buffer, and applied to the TSK
BioAssist Q column. The ORF2 protein was eluted with a gradient of
0-1000 mM NaCl. Active fractions were pooled, dialyzed
against Buffer A, and applied to the same column. After three times of
column chromatography, the purified ORF2 protein was obtained.
Enzyme Activities--
Methyltransferase activities were
measured according to the method of Raha et al. (25)
with a slight modification. The reaction mixture consisted of 25 µl
of 25 mM substrate (methyl acceptor), 25 µl of buffer B
(125 mM Tris-Cl, pH 8.8, 2 mM
2-mercaptoethanol, 50 µM MgCl2, 160 µM EDTA), 25 µl of 4 mM
S-adenosyl-L-methionine (AdoMet) (45 nCi of
S-adenosyl-L-[methyl-14C]methionine),
and 25 µl of methyltransferase. The reaction was started by the
addition of enzyme. After incubation for 30 min at 37 °C, the
reaction was stopped by addition of 75 µl of charcoal suspension (133 g/liter in 0.1 M acetic acid), which adsorbs the remained
S-adenosyl-L-[methyl-14C]methionine,
and incubated on ice for 10 min. After centrifugation for 10 min, 75 µl of supernatant was removed for assay, and the amount of
14C in methyl acceptors was measured with a liquid
scintillation counter (model 3200C, Aloka Instruments Co., Tokyo,
Japan). The enzyme activity was calculated as nanomoles of methyl
groups transferred per min. The pH in the reaction mixture was adjusted
by the following buffers, 125 mM of potassium phosphate (pH
6.0-7.0), 125 mM Hepes-KOH (pH 7.0-8.0), 125 mM Tris-HCl (pH 7.5-8.8), and 125 mM
bicarbonate (pH 8.5-10.5). For the measurements of
Km and Vmax, the concentrations of methyl acceptors (glycine, sarcosine, and
dimethylglycine) were changed between 0 and 50 mM. Methyl
donor, AdoMet, was changed between 0 and 10 mM. The
reaction products of methyltransferases were identified by two methods,
thin layer chromatography and time of flight mass spectroscopy (model
KOMPACT MALDI IV tDE, Shimadzu/Kratos) (26). For thin layer
chromatography, silica gel 60 (Merck) and phenol/water (80:20, v/v)
solvent system were used. The radioactive spots were detected by
autoradiography, and the authentic standards were revealed
colorimetrically. The orRf values for glycine, sarcosine,
dimethylglycine, and betaine were 0.23, 0.47, 0.60, and 0.67, respectively.
Chemical Modifications of ORF1 and ORF2 with
Phenylglyoxal--
ORF1 and ORF2 proteins (0.5 mg/ml) were
incubated with 0, 2, 5, and 10 mM phenylglyoxal in 20 mM Tris-HCl buffer (pH 8.8) at 37 °C. After 60 min, an
aliquot (10 µl) of the reaction mixture was added to 2.0 ml of the
assay mixture, and the residual enzyme activity was determined as above.
Salt Upshock and Downshock of A. halophytica Cells--
A.
halophyticacells were grown for 14 days in the growth medium
described above, containing 0.5 or 2.5 M NaCl. The cells
were transferred to the new medium containing 2.5 or 0.5 M
NaCl, respectively. After appropriate times, cells were harvested and
used for the Western blotting experiments.
Other Methods--
SDS-PAGE and Western blotting analysis was
carried out according to standard protocol (22). Antibodies raised
against the ORF1 and ORF2 proteins were prepared by injection of
purified ORF1 and ORF2 proteins into white New Zealand female rabbits, respectively (26). Protein concentrations were determined by Lowry's
method. The molecular weights of native ORF1 and ORF2 proteins were
estimated by a Superdex 200 column (60 × 2.6 cm) gel filtration
column chromatography (Amersham Biosciences). Glycinebetaine content
was analyzed by time of flight mass spectroscopy as described previously (26).
Cloning of Methyltransferase Genes from A. halophytica--
During
the genome sequencing of A. halophytica, two ORFs (ORF1 and
ORF2) homologous to the EcGSMT and EcSDMT were found. The predicted
gene products for ORF1 and ORF2 consist of 265 amino acids with a
molecular mass of 31,211 Da and 277 amino acids with a molecular mass
of 31,611 Da, respectively (Fig.
1A). The homology search
revealed that a protein encoded by ORF1 is highly homologous to the
EcGSMT (~66% identity in amino acids) and N-terminal part of
heterotrophic eubacterium A. halophila methyltransferase
(~61% identity in amino acids). The ORF1 protein showed
some homology to the rat glycine N-methyltransferase
(~35% identity in amino acids) but essentially no
homology to other methyltransferases including phosphoethanolamine
N-methyltransferase (27). The protein encoded by ORF2 showed
the highest homology to EcSDMT (~52% identity in amino
acids) and C-terminal part of A. halophila methyltransferase
(~49% identity in amino acids). No other homologous sequence was found. Among small molecule methyltransferases, consensus sequences for AdoMet-binding domains (motifs I, post I, II, and III)
have been reported (28). As shown in Fig. 1A, the putative AdoMet-binding motifs were found in both ORF1 and ORF2 proteins although identification of post I, II, and III motifs were somewhat obscure.
Expression and Purification of the Cloned Genes in E. coli--
The ORF1 and ORF2 were expressed in E. coli BL21
(DE3) under the control of the T7 promoter. It was found that both ORF1
and ORF2 proteins were expressed at high levels as soluble proteins (Fig. 2A). The SDS-PAGE
suggested that molecular masses for ORF1 and ORF2 proteins were 33 and
31 kDa, respectively, which are in good agreement with the values
calculated from the nucleotide sequences. Both proteins were purified
to homogeneity by ammonium sulfate precipitation and ion-exchange
column chromatography. Molecular masses of native enzymes for ORF1 and
ORF2 were determined by gel filtration chromatography as 32 and 29 kDa,
respectively (Fig. 2B). A part of the ORF1 protein was
purified as a tetramer, but it was inactive (data not shown). These
results indicate that both enzymes are monomer, which is the same to
EcGSMT and EcSDMT (16) but different from the mammalian GMTs (18),
which are tetramer.
Substrate Specificities and Kinetic Properties for ORF1 and ORF2
Proteins--
We tested whether the ORF1 and ORF2 enzymes could
synthesize betaine from glycine. AdoMet acts as methyl donor in both
enzymes. The reaction products of ORF1 and ORF2 enzymes were analyzed
by thin layer chromatography and time of flight mass spectroscopy as
described under "Materials and Methods." It was found that the ORF1
protein catalyzed the methyl transfer to glycine and sarcosine but not
to dimethylglycine (Fig. 3A).
By contrast, the ORF2 protein catalyzed the methyl transfer to
dimethylglycine but not to glycine and sarcosine. Neither ethanolamine
or ethanolamine derivatives were N-methylated by both ORF1
and ORF2. None of tested amino acids, except glycine, were
N-methylated. Acids such as isovaleric-, n-butyric-,
propionic-, and t-butylacetic-acid were also inactive as acceptors of
methyl group. These results indicate that the ORF1 specifically
catalyzed the methylation reactions of glycine and sarcosine, whereas
the ORF2 catalyzed the methylation of dimethylglycine, and therefore,
betaine synthesis from glycine is possible if two enzymes work
together. The ORF1 and ORF2 were designated as ApGSMT and ApDMT,
respectively.
Kinetic parameters for the methyl transfer reactions by ORF1 and ORF2
were examined. The apparent kinetic parameters for substrates were
determined with other substrates present in excess. Both enzymes
displayed Michaelis-Menten kinetics for their substrates (data not
shown). Fig. 3B shows that the apparent
Km value of ORF1 was significantly smaller for
glycine (1.5 mM) than those of EcGSMT (18 mM)
and human GMT (6.3 mM) but higher than that of rat GMT
(0.44 mM). The apparent Km values of ORF1 for sarcosine (0.8 mM) and of ORF2 for dimethylglycine
(0.8 mM) were also smaller than those of EcGSMT (2.3 mM) and EcSDMT (6.1 mM) for sarcosine and of
EcSDMT (4.9 mM) for dimethylglycine. By contrast, the
apparent Km values of ORF1 and ORF2 for AdoMet were
similar or slightly larger than those of EcGSMT, EcSDMT, and mammalian
GMTs. The Vmax values of ORF1 and ORF2 were similar or lower than those of EcGSMT and EcSDMT, respectively. As a
whole, the results indicate that the affinities for the methyl acceptors were higher in A. halophytica than those in
E. halochloris, whereas the affinities for the
methyl donor and Vmax were similar between them.
Inhibitors for ApGSMT and ApDMTs--
Inhibitors for ApGSMT (ORF1)
and ApDMT (ORF2) were examined. As shown in Fig.
4, acetate inhibited the methylation
reactions of glycine and sarcosine by ApGSMT about 78 and 62%,
respectively. Dimethylglycine also inhibited these reactions about 67 and 39%, respectively. Other amino acids and ethanolamine derivatives
did not inhibit or only slightly inhibited the activity of ApGSMT. The
methyl transfer reaction of dimethylglycine by ApDMT was moderately inhibited by n-butylic acid. However, amino acids including glycine and
acids such as t-butylic-, isovaleric-, and propionic-acid essentially
did not inhibit its reaction. These results indicate that acetate and
dimethylglycine were inhibitors for ApGSMT and n-butylic acid was a
inhibitor for ApDMT.
It is known that AdoMet-dependent methyltransferases are
strongly inhibited by the reaction product
S-adenosyl-L-homocysteine (AdoHcy) (29). Fig.
5A shows that the activities
of ApGSMT and ApDMT were also inhibited by AdoHcy. However, the
Ki values for ApGSMT and ApDMT were in the range of
0.3-0.6 mM, which were significantly larger than those of
mammalian GMTs, 0.035-0.08 mM (17), and other
methyltransferases (e.g. 0.4 µM for tRNA methyltransferase) (29). As shown in Fig. 5B, a final
product, betaine, did not inhibit the methyl transfer reactions of
ApGSMT and ApDMT at all. These results indicate that ApGSMT and ApDMT were weakly inhibited by the reaction products, AdoHcy and betaine, which might be suitable for the accumulation of high
concentrations.
Effects of pH Activities Profiles and Salts on the Activities of
ApGSMT and ApDMTs--
The pH activity profiles of ApGSMT and ApDMT
are shown in Fig. 6A. The
maximal activities for both enzymes were around pH 8.8. The activities
were remained high at more alkaline pH but decreased sharply at the
acidic side of the optimal pH.
NaCl and KCl were two important salts for A. halophytica
(14, 20). NaCl and KCl inhibited the methylation reactions of ApGSMT
and ApDMT with different patterns (Fig. 6, B and
C). At 1.0 M NaCl, methyl transfers to glycine
by ApGSMT and to dimethylglycine by ApDMT were inhibited only about
17%, whereas the methyl transfer to sarcosine by ApGSMT was inhibited
about 70% (Fig. 6B). By contrast, KCl inhibited the methyl
transfer reactions by ApGSMT and ApDMT to the similar extends, about
50% at 1.0 M KCl (Fig. 6C). These results
indicate that the activities of ApGSMT and ApDMT were affected
moderately by NaCl and KCl although the methylation of sarcosine by
ApGSMT was inhibited considerably.
Salt-induced Increase of Methyl Transfer Activities and Enzyme
Levels of ApGSMT and ApDMT in A. halophytica Cells--
Although the
above results strongly suggest that A. halophytica cells
synthesize betaine from glycine by a three step methylation, we
investigated whether the glycine-methylation pathway operates in
A. halophytica cells. The results shown in Fig.
7A clearly indicate that
A. halophytica cells have methyltransferase activities of
glycine, sarcosine, and dimethylglycine using AdoMet as a methyl donor.
All methyltransferase activities increased about 1.6-2.5-fold upon the
increase of NaCl from 0.5 M to 2.5 M. By
contrast, the methyltransferase activities of glycine, sarcosine, and
dimethylglycine almost could not be detected in
Synechococcus sp. PCC 7942 cells.
Western blotting experiments showed that the accumulation levels of
ApGSMT and ApDMT increased upon the increase of NaCl in growth medium
from 0.5 M to 2.5 M and decreased upon the
decrease of NaCl from 2.5 M to 0.5 M (Fig.
7B). These results support the viewpoint that A. halophytica cells synthesize betaine from glycine by a three-step
methylation and that betaine levels are regulated by salts in
concomitant with the levels of ApGSMT and ApDMT.
Co-expression of ApGSMT and ApDMT in E. coli Cells--
To
investigate whether ApGSMT and ApDMT could synthesize betaine in
E. coli, the ApGSMT and ApDMT were co-expressed in BL21 cells. The control cells could synthesize betaine by the E. coli betaine synthesis genes (choline dehydrogenase and
betaine-aldehyde dehydrogenase). As shown in Fig.
8, it was found that the E. coli (BL21) cells co-expressing ApGSMT and ApDMT grew faster than
the control cells. Upon the increase of salinity, the growth rates of
both cells decreased, but the growth rates of co-expressing cells were
always higher than the control cells. Accumulation levels of betaine in
the co-expressing cells were 3- to 5.5-fold higher than those of
control cells. These results suggest that the co-expression of ApGSMT
and ApDMT could synthesize betaine about 2- to 4.5-fold more than the
E. coli betaine synthesis genes, which caused the enhanced
growth rate of transformed E. coli cells.
Site-directed Mutagenesis of Arg-169 in ApGSMT--
Among the many
AdoMet-dependent methyltransferases, the consensus
sequences on AdoMet-binding domains have been proposed (28, 29). But,
the structural information on methyl acceptors are largely unknown.
Previously, no information was available on the amino acids of
methyltransferases involved in betaine synthesis. Here, we focused on
the methyl acceptor binding domains. Fortunately, Arg-176 in rat GMT
has been proposed as a possible glycine binding site (17). It was
found that several amino acids around Arg-176 in rat GMT are conserved
among four methyltransferases (Fig. 1). To test the role of Arg for
substrate binding in ApGSMT, a chemical modification study of Arg was
carried out by using phenylglyoxal. When ApGSMT was incubated with
phenylglyoxal as described under "Materials and Methods," the
glycine methyltransferase activity decreased. At 10 mM
phenylglyoxal, the activity decreased to 23% of the control activity
(data not shown). By contrast, the inhibition of dimethylglycine
methyltransferase activities of ApDMT by phenylglyoxal was negligible.
These data suggest the importance of Arg for methyltransferase in
ApGSMT but not in ApDMT. To test more directly, the Arg-169 in
ApGSMT was modified to Lys and Glu by the site-directed mutagenesis (Fig. 9). The mutants, R169K and R169E,
were expressed in E. coli at similar levels to wild type and
purified as soluble proteins. It was found that the R169E mutant was
completely inactive (Fig. 9). By contrast, the
Vmax values of R169K were about 10-20% of the
wild-type enzyme when glycine and sarcosine were used as methyl group
acceptors. The mutant R169K could not catalyze the methylation of
dimethylglycine. The apparent Km values of R169K for glycine and sarcosine increased about 50- and 63-fold, respectively, whereas the apparent Km values for AdoMet changed
only within 2-fold. These results suggest that the Arg-169 in ApGSMT was involved in binding of glycine and sarcosine but not of AdoMet.
Site-directed Mutagenesis of Pro-171 and Met-172 in
ApDMT--
Substrate specificity of ApDMT was unique, and we have
no homologous enzyme that could be compared with it based on structural information. However, it was found that five amino acids after motif
III, FTDPM, were conserved among three methyltransferases as shown in
Figs. 1 and 10. Therefore, we tested
the role of Pro-171 and Met-172 as a possible methyl group binding site
in ApDMT. Three mutants, P171Q, M172R, and P171Q/M172R, were
constructed, expressed in E. coli, and purified as soluble
proteins. As shown in Fig. 10, the mutants, M172R and P171Q/M172R, were
almost inactive. By contrast, the Vmax value of
P172Q was about 27% of the wild-type enzyme when dimethylglycine was
used as a methyl group acceptor. Glycine and sarcosine were ineffective
as methyl group acceptors. The apparent Km value for
dimethylglycine increased about 15-fold, but the apparent
Km value for AdoMet changed only within 2-fold.
These results suggest that the Pro-171 and Met-172 in ApDMT were
involved in binding of dimethylglycine.
Present results clearly indicate that in contrast to other
oxygen-evolving photosynthetic organisms, betaine is synthesized from
glycine by a three-step methylation in a halotolerant cyanobacterium A. halophytica. This conclusion was supported by several
experimental evidences. Isolated genes exhibited homology to recently
discovered glycine methyltransferase genes (Fig. 1) (15, 16). The
purified enzymes catalyzed the methyl transfer reactions to glycine,
sarcosine, and dimethylglycine (Fig. 3). The methyl transfer activities
of glycine, sarcosine, and dimethylglycine were detected in A. halophytica but not in Synechococcus sp. PCC 7942, a
betaine non-accumulating cyanobacterium (Fig. 7A). The
accumulation levels of ApGSMT and ApDMT in A. halophytica
cells increased upon the increase of salinity (Fig. 7B).
Co-expression of ApGSMT and ApDMT in E. coli cells increased
the accumulation levels of betaine (Fig. 8).
Two methyltransferase genes, encoding ApGSMT and ApDMT, were isolated
from A. halophytica (Fig. 1). ApGSMT catalyzed the
methylation reactions of glycine and sarcosine, whereas ApDMT
specifically catalyzed the methylation reaction of dimethylglycine
(Fig. 3). Homologous sequences to ApGSMT and ApDMT were only found in
E. halochloris and A. halophila. A. halophytica
methyltransferases were different from that of A. halophila
in which one gene was found (15). ApGSMT and ApDMT of A. halophytica were similar to EcGSMT and EcSDMT of E. halochloris, respectively, but substrate specificity of ApDMT was
different from EcSDMT (Fig. 3).
Similar to betaine (trimethylglycine), choline is a trimethyl compound
of ethanolamine (EA), and synthesized from EA via free-base, phospho-base, and phosphatidyl (Ptd)-base, depending on the species (1,
27). Several N-methyltransferases are involved in choline biogenesis. For instance, three-step methylation reactions of phosphatidylethanolamine (Ptd-EA), Ptd-monomethyl-EA, and
Ptd-dimethyl-EA have been catalyzed by a single Ptd-EA
N-methyltransferase in the cases of Rhodobacter
sphaeroides and liver (30, 31). However, in yeast, these reactions
are catalyzed by two enzymes, one mediating the first methylation of
Ptd-EA and another mediating the last two (32). In the phospho-base
pathway, presence of three separate N-methyltransferases has
been reported in nerve tissues (33), whereas in plants, three-step
methylation reactions were catalyzed by a single enzyme comprising two
similar tandem methyltransferase domains (27). Thus, the biogenesis of
N-trimethyl compounds occurs via diversity
methyltransferases. Despite of diversity methyltransferases, betaine
synthesis from glycine was only observed in halotolerant organisms
(15). This suggests that the methyltransferase genes, encoding ApGSMT
and ApDMT, in A. halophytica might evolve independently from
those of methyltransferases in choline biogenesis and mammalian glycine
N-methyltransferase.
Fig. 4 shows that acetate inhibited the methyl transfers to glycine and
sarcosine. The inhibition of glycine methylation by acetate in
mammalian GMTs has been reported (17, 18). These results suggest some
similarity of methyl acceptor domain between ApGSMT and mammalian GMT
although mammalian GMTs could not catalyze the methyl transfer to
sarcosine. Mammalian GMTs are tetrameric enzyme (17, 18), whereas
ApGSMT was monomer. It has also been reported that rat GMT plays
another function such as a major folate-binding protein (34). Despite
these functional differences between ApGSMT and mammalian GMTs, it was
found that the amino acid sequence after AdoMet binding motif III was
relatively conserved between them (Fig. 9). Especially Arg-176 in rat
GMT, the putative glycine binding site in rat GMT, was conserved. The
chemical modification of Arg by phenylglyoxal and site-directed
mutagenesis experiments (Fig. 9) showed that the Arg-169 in ApGSMT
plays an important role for binding of glycine and sarcosine but not of
AdoMet.
ApDMT is an unique enzyme. Its substrate specificity was different from
that of EcSDMT (Fig. 3). The amino acid sequence after AdoMet binding
motif III in ApDMT did not show the homology to that of mammalian GMTs.
Site-directed mutagenesis of Pro-170 and Met-171 showed that these
amino acids play important role for binding of dimethylglycine, but not
of glycine or sarcosine. The results of Figs. 9 and 10 suggest that the
amino acid sequence just after AdoMet binding motif III might play
important role for binding of methyl acceptor group in small molecule
N-methyltransferases, which remains to be tested experimentally.
AdoHcy is a potent inhibitor in almost all AdoMet-dependent
methyltransferases. For the continuous synthesis of betaine, AdoHcy must be removed by AdoHcy-hydrolase, which catalyzes both synthesis and
hydrolysis reactions. The results of Fig. 5A show that in contrast to most other AdoMet-dependent methyltransferases,
the activities of ApGSMT and ApDMT were inhibited by AdoHcy with
relatively low affinities. The Ki values of ApGSMT
and ApDMT were about 10-fold higher than that of rat GMT and 1000-fold
higher than that of tRNA methyltransferase (Fig. 5A).
Allowance of relatively high concentrations of AdoHcy during the
betaine synthesis could shift the equilibrium to the hydrolysis
direction that would be suitable for the synthesis of large amount of betaine.
Fig. 5B shows that one of final product, betaine, did not
inhibit the methyl transfer activities of ApGSMT and ApDMT. This property is also suitable for the synthesis of high concentrations of
betaine. The results of Fig. 4 indicate that acetate and
dimethylglycine are inhibitors for ApGSMT whereas n-butylic acid was an
inhibitor for ApDMT. Any examined amino acids except glycine,
ethanolamines derivatives, and several acids did not affect the methyl
transfer activities. These facts suggest that betaine synthesis could
occur relatively without interference of metabolites.
Hitherto, extensive studies have been carried out for genetic
engineering of betaine accumulation (35, 36). All of them used the
genes encoding choline-oxidizing enzymes such as choline monooxygenase,
choline dehydrogenase, choline oxidase, and betaine-aldehyde dehydrogenase. However, the produced betaine levels were generally low.
Several factors limiting the betaine levels are considered. Supply of
choline, transport of choline to the place choline oxidizing enzyme
localized, and increase of choline-oxidizing enzyme levels might be
important to improve the abiotic tolerance of plants. The data of Fig.
8 show that ApGSMT and ApDMT could produce large amount of betaine in
E. coli. Therefore, overexpression of ApGSMT and ApDMT in
photosynthetic organisms are interesting subjects to be tested, which
is in progress in this laboratory.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
betaine aldehyde
glycine betaine. The enzyme
involved in the second step seems to be the same in plants, animals,
and bacteria, namely NAD+-dependent
betaine-aldehyde dehydrogenase (4-6). By contrast, different enzymes
are involved for the first step. In plants, it was catalyzed by a novel
Rieske-type iron-sulfur enzyme choline monooxygenase (7, 8). In animals
and many bacteria, the first step is catalyzed by membrane-bound
choline dehydrogenase or soluble choline oxidase (9-11). In some
bacteria, choline dehydrogenase and choline oxidase also catalyze the
second oxidation step (9-11).
MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and E. coli BL21(DE3) cells were grown at 37 °C in LB medium or M9
minimal medium supplemented with 0.2% glucose as a sole carbon source.
Ampicillin was used at the final concentration of 50 µg/ml. A. halophytica cells were grown photoautotrophically in BG11 liquid
medium plus 18 mM NaNO3 and Turk Island salt
solution at 28 °C as previously described (19, 21). TSK BioAssist Q column was purchased from Tosoh Co. (Tokyo, Japan). Radiolabeled S-adenosyl-L-[methyl-14C]methionine
was purchased from Amersham Biosciences.
. The DNA sequence of ORF1 and ORF2 in plasmids was
determined using a 310-genetic analyzer (Applied Biosystems, Foster
City, CA) and analyzed with the DNASIS program (Hitachi Software
Engineering Co., Kanagawa, Japan).
Primers for isolation and expression of ORF1 and ORF2 genes
-D-thiogalactopyranoside (IPTG)
was added, and the cells were grown a further 3 h. The cells were
harvested by centrifugation at 5,000 × g for 10 min and washed twice with Buffer A (20 mM Tris-Cl, pH 8.0, containing 2 mM 2-mercaptoethanol). Total cell extracts
were obtained by sonication with Kubota Insonator 201 M at
180 W for 15 min. Unbroken cells were removed by centrifugation at
10,000 × g for 10 min.
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Fig. 1.
Alignment of the deduced amino acid sequences
and phylogenetic analysis of seven methyltransferases.
A, alignment of the deduced amino acid sequences of ORF1.
The sequences were aligned by the program ClustalW. The amino acid
residues conserved in all sequences are star and
conservative substitutions are shown by dot. Putative AdoMet
binding motifs I, post I, II, and III were marked above the alignment.
The mutated amino acid, Arg (R), was also marked above the
alignment. AcGSDMT, A. halophila methyltransferase
(15). B, alignment of the deduced amino acid sequences of
ORF2. The mutated amino acids, Pro (P) and Met
(M), were also marked above the alignment. C,
phylogenetic analysis of seven methyltransferases. Multiple sequence
alignment and generation of phylogenetic tree were performed with
ClustalW and TreeView software, respectively.
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Fig. 2.
SDS-PAGE and gel filtration chromatography of
ORF1 and ORF2 proteins. ORF1 and ORF2 proteins were expressed in
E. coli BL21 cells using pET vectors. A, SDS-PAGE
of ORF1 and ORF2 proteins expressed in E. coli. Lane
1, soluble fraction of control cells; lane 2, molecular
weight marker; lane 3, soluble fraction of E. coli cells expressing ORF1 protein; lane 4, soluble
fraction of E. coli cells expressing ORF2 protein;
lane 5, purified ORF1; lane 6, purified ORF2
protein. B, estimation of molecular masses of ORF1 and ORF2
proteins by gel filtration chromatography. Aldolase (158 kDa), albumin
(67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and
ribonuclease A (13.7 kDa) were used as molecular mass
markers.
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Fig. 3.
Substrate specificity and kinetic parameters
for ORF1 and ORF2 proteins. A, substrate specificity.
Activities were measured at pH 8.8 and represented by relative values.
The concentration of AdoMet was 1 mM. The concentration of
methyl group acceptors was 25 mM. B, kinetic
parameters. The apparent kinetic parameters for substrates were
determined with other substrates present in excess. The concentrations
of fixed substrates were 1 mM for AdoMet and 25 mM for methyl group acceptors. Each value shows the average
of three independent measurements (S.E. was within 15%).
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Fig. 4.
Inhibitors for the ApGSMT and ApDMT.
Effects of inhibitors on the activities were measured at pH 8.8. The
concentrations of AdoMet and methyl group acceptors were 1 and 25 mM, respectively. The concentration of inhibitors was 250 mM. Each value shows the average of three independent
measurements.
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Fig. 5.
Effects of AdoHcy and betaine on the
activities of ApGSMT and ApDMT. The concentrations of AdoHcy and
betaine were changed as described. Other experimental conditions were
the same as those in Fig. 4. Each value shows the average of three
independent measurements.
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Fig. 6.
Effects of pH and salts on the activities of
ApGSMT and ApDMT. A, effects of pH. B,
effects of NaCl. C, effects of KCl. The concentrations of
salts and pH were changed as described under "Materials and
Methods." Other experimental conditions were the same as those in
Fig. 4. Each value shows the average of three independent
measurements.
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Fig. 7.
Methyltransferase activities and accumulation
levels of ApGSMT and ApDMT in A. halophytica cells.
A, methyltransferase activities in A. halophytica
cells. Cell extracts were prepared as described under "Materials and
Methods." Experimental conditions for the activity measurements were
the same as those in Fig. 4. Each value shows the average of three
independent measurements. B, immunoblotting of ApGSMT and
ApDMT upon upshock and downshock. For the upshock experiments, the
concentration of NaCl in growth medium was changed from 0.5 to 2.5 M. For the downshock experiments, the concentration of NaCl
was changed from 2.5 to 0.5 M.
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Fig. 8.
Growth rates and accumulation levels of
betaine in E. coli cells co-expressing ApGSMT and
ApDMT. A-D, growth rates of E. coli cells
co-expressing ApGSMT and ApDMT. E. coli (BL21) cells were
grown in LB medium. IPTG was added when OD620 reached 0.5. A, LB; B, LB + 0.1 M NaCl;
C, LB + 0.3 M NaCl; D, LB + 0.5 M NaCl. Circle, ApGSMT and ApDMT co-expressing
cells; square, control cells. E, accumulation
levels of betaine. Cells were harvested at 3 h after addition of
IPTG. Each value shows the average of three independent
measurements.
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Fig. 9.
Effects of mutations of Arg-169 on the
activities of ApGSMT and ApDMT. Kinetic parameters for ApGSMT
mutants R169K and R169E. Kinetic parameters were measured as those in
Fig. 3. Each value shows the average of three independent measurements
(S.E. was within 15%).
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Fig. 10.
Effects of mutations of Pro-171 and Met-172
on the activities of ApDMT. A, relative activities for
ApDMT mutants P171Q, M172R, and P171Q/M172R. B, kinetic
parameters for the P171Q mutant of ApDMT. Kinetic parameters were
measured as those in Fig. 3. Each value shows the average of three
independent measurements (S.E. was within 15%).
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Eiko Tsunekawa for expert technical assistance.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education and Science and Culture of Japan, the High-Tech Research Center of Meijo University.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 DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB094497 and AB094498.
** To whom correspondence should be addressed: Research Institute of Meijo University, Tenpaku-ku, Nagoya, Aichi 468-8502, Japan. Tel.: 81-52-832-1151; Fax: 81-52-832-1545; E-mail: takabe@ccmfs.meijo-u.ac.jp.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M210970200
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ABBREVIATIONS |
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The abbreviations used are:
EcGSMT, E.
halochloris glycine sarcosine methyltransferase;
EcSDMT, E.
halochloris sarcosine dimethylglycine methyltransferase;
GMT, glycine N-methyltransferase;
IPTG, isopropyl-1-thio--D-galactopyranoside;
AdoMet, S-adenosyl-L-methionine;
AdoHcy, S-adenosyl-L-homocysteine;
ApDMT, A.
halophytica dimethylglycine methyltransferase;
ApGSMT, A. halophytica glycine sarcosine methyltransferase;
EA, ethanolamine;
Ptd, phosphatidyl;
Ptd-EA, phosphatidylethanolamine.
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