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Journal of Bacteriology, December 2000, p. 7078-7082, Vol. 182, No. 24
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of GTPase Activity of TrmE, a
Member of a Novel GTPase Superfamily, from Thermotoga
maritima
Kunitoshi
Yamanaka,
Jihwan
Hwang, and
Masayori
Inouye*
Department of Biochemistry, Robert Wood
Johnson Medical School, Piscataway, New Jersey 08854
Received 19 May 2000/Accepted 7 August 2000
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ABSTRACT |
A gene encoding a putative GTP-binding protein, a TrmE homologue
that is highly conserved in both prokaryotes and eukaryotes, was cloned
from Thermotoga maritima, a hyperthermophilic bacterium. T. maritima TrmE was overexpressed in Escherichia
coli and purified. TrmE has a GTPase activity but no ATPase
activity. The GTPase activity can be competed with GTP, GDP, and
dGTP but not with GMP, ATP, CTP, or UTP. Km and
kcat at 70°C were 833 µM and 9.3 min
1, respectively. Our results indicate that TrmE is a
GTP-binding protein with a very high intrinsic GTP hydrolysis rate. We
also propose that TrmE homologues constitute a novel subfamily of the GTPase superfamily.
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TEXT |
Proteins possessing GTP-binding and
GTPase activities play essential roles in cell proliferation in
both prokaryotes and eukaryotes. Their functions are very diversified
and include involvement in protein translation (e.g., EF-Tu), signal
transduction (e.g., small G proteins), cell growth (e.g., RAS),
vesicular transport (e.g., RAB), and protein translocation across
membranes (e.g., SRP), etc. (3, 4, 14). They all contain
well-conserved motifs
G-1, G-3, and G-4which are important for
GTP-binding activity (4). Another motif, G-2, is not
involved in binding to GTP but is involved in interaction with an
effector molecule (3, 4). G-2 sequences are well conserved
within each subfamily in the large GTPase family. Most GTP-binding
proteins possess a very high intrinsic GTP hydrolysis rate, while their
GTPase activities are highly stimulated by effector molecules
through their binding to the G-2 motif. In general, the ratio of the
active GTP-bound form to the inactive GDP-bound form of a GTP-binding protein is crucial for its functional regulation, and this process includes GTP hydrolysis that results from conformational changes of the
GTP-binding protein (14).
Analysis of the Escherichia coli genome revealed that it
contains a GTP-binding protein encoded by the gene originally
designated thdF (1, 5). Recently, this gene was
shown to be essential in E. coli and involved in tRNA
modification and was thus redesignated trmE (6).
The E. coli TrmE protein contains plausible GTP-binding motifs G-1 to G-4 consisting of 454 amino acid residues (6). To date, more than 20 proteins homologous to TrmE have been found in
prokaryotes and eukaryotes (Fig. 1).
Notably, all eubacteria, but not archaea, whose entire genomes have
been sequenced contain TrmE; in addition, a TrmE homologue exists in
expressed sequence tags from humans (data not shown). A TrmE homologue
in Saccharomyces cerevisiae, MSS1, has been proposed to be a
GTPase and is thought to be involved in translational regulation in
mitochondria (9, 10). In this study we cloned the
trmE gene, highly homologous to the E. coli trmE
gene, from the hyperthermophilic bacterium Thermotoga
maritima (19), and its product was highly purified. Its
biochemical characterization revealed that T. maritima TrmE is indeed a GTP-binding protein having a very high intrinsic GTPase activity at 80°C.
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FIG. 1.
Amino acid sequence alignments of TrmE
homologues. A homology search was carried out by the BLAST program
(2). Residues identical to those of T. maritima TrmE are shown as dots, and gaps are indicated by dashes.
The GTP-binding motifs G-1 to G-4 and well-conserved regions I to IV
are indicated by bars above the sequences. Tm, T. maritima
(National Center for Biotechnology Information [NCBI] protein
database accession no. AAD35356.1); Ae, Aquifex aeolicus
(AAC06992.1); Bs, Bacillus subtilis (CAA44403.1); Ec,
E. coli (AAC76729); Hi, Haemophilus
influenzae (AAC22664.1); Pp, Pseudomonas putida
(CAA44418.1); Ct, Chlamydia trachomatis (AAC68293.1); Mp,
Mycoplasma pneumoniae (AAB95794); Rp, Rickettsia
prowazekii (CAA15187); Ss, Synechocystis sp. strain
PCC6803 (BAA17896); Se, Synechococcus elongatus
(CAB46651.1); Cc, Cyanidium caldarium (chloroplast genome)
(AAF12952); Sc, S. cerevisiae (CAA49238.1); Sp,
Schizosaccharomyces pombe (CAB60697).
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The trmE gene encodes a GTP-binding protein.
GTP-binding motifs G-1 to G-4 are well conserved in all TrmE homologues
(Fig. 1), strongly indicating that T. maritima TrmE is a
GTP-binding protein and possesses a GTP hydrolysis activity. Motif G-2
is thought to be involved in binding to an effector molecule but not in
GTP binding. The consensus sequence for G-2 is limited to TrmE
homologues and cannot be applied to other subfamilies of GTP-binding
proteins (data not shown), suggesting that TrmE homologues constitute a
novel subfamily of the GTPase superfamily and that TrmE probably
interacts with a specific effector molecule, which could regulate the
GTPase activity of TrmE. The GTP-binding motifs are located within
the third quarter of this protein from the N-terminal end (Fig. 1).
Besides GTP-binding motifs, at least four well-conserved regions
(regions I, II, III, and IV) can be assigned (Fig.
1). Although
their
functional significance is unknown at present, these regions
are likely
to be related to the TrmE-specific function. Only the
GTP-binding
domains G-1 to G-4 are involved in GTP binding and
GTPase activity
in
E. coli TrmE (
6); therefore, regions I to
IV
are unlikely to be involved in GTP binding and GTPase activity
in
T. maritima. It is, however, possible that they are
associated
with the regulation of GTP binding and GTPase activity
and/or
that they might be involved in tRNA modification, since
trmE mutants
exhibit deficiency in biosynthesis of
5-methylaminomethyl-2-thiouridine
of tRNA (
11). As three
E. coli GTPases
Ffh, EF-G, and Era
are
known to bind
to RNA (
8,
12,
13,
15,
17,
18,
21,
22) and their RNA binding
is closely associated with their GTPase
activities, the TrmE
GTPase may also be closely associated with
its tRNA modification
activity. It is interesting that the C-terminal
sequence CVGK in region
IV seems to be a consensus sequence, CAAX,
where A represents an
aliphatic amino acid residue and X represents
any amino acid residue,
for isoprenylation in the Ras protein
(
23), although no
isoprenylation has been reported and no genes
involved in
isoprenylation have been identified in the prokaryotes
so far. It is
also interesting that
E. coli TrmE has been shown
to be
localized in both the cytoplasm and the inner membrane (
6).
During the process of a homology search for TrmE, we found another
homologous protein, which has been registered as TM1446
in the
T. maritima genome (
19). This protein contains two tandem
repeats of the GTP-binding domain of TrmE, but its homologues,
unlike
those of TrmE, are found only in the prokaryotes (data
not
shown).
Purification of T. maritima TrmE.
To clone the
trmE gene from T. maritima, the genomic DNA of
T. maritima (a generous gift from Francis E. Jenney, Jr.,
University of Georgia) was used as a template for PCR. Primers 9541 (5'-AGACAACATATGGATACCATTGTCGCTGTAG-3' [an
NdeI site is underlined]) and 9540 (5'-CCAAGCTTTCATTTTCCAACGCAGAAA-3' [a
HindIII site is underlined]) were used. PCR was carried
out with 30 cycles of amplification of 1 min at 95°C, 2 min at
50°C, and 2 min at 72°C. The PCR product was digested with
NdeI and HindIII and cloned into the
NdeI-HindIII site of pET17b, yielding pET-TmTrmE. The DNA sequences of the inserted fragment were confirmed.
Plasmid pET-TmTrmE was introduced into
E. coli BL21(DE3)
cells containing the
ndk::Cm
r mutation
(
16). The
ndk disruption strain was used to avoid
contamination of GTPase with nucleoside diphosphate kinase in
the
T. maritima TrmE preparation. Transformed cells were grown
at 37°C to mid-exponential phase in 3 liters of M9 medium
supplemented
with 0.2% Casamino Acids and 50 µg of ampicillin per
ml. TrmE
was induced for 5 h in the presence of 1 mM
isopropyl-
-thiogalactopyranoside.
TrmE was well expressed as
approximately 40% of the total cellular
protein (data not shown). The
cells were harvested by centrifugation,
washed with 10 mM bis-Tris
buffer (pH 7.0), and resuspended in
45 ml of the same buffer. The cells
were broken by two passes
through a French press at 9,000 lb/in
2, followed by centrifugation at 8,000 ×
g for 10 min to remove
cell debris and unbroken cells and by
ultracentrifugation at 100,000
×
g for 1 h to
remove membrane and insoluble fractions. The soluble
fraction was
treated at 70°C for 10 min to denature
E. coli proteins
and centrifuged at 12,000 ×
g for 20 min to remove
denatured proteins.
Ammonium sulfate was then added to the resulting
soluble fraction
to 80% saturation. The solution was kept on ice for
1 h and centrifuged
at 16,000 ×
g for 20 min to
obtain the
precipitates.
The ammonium sulfate precipitate was solubilized in 20 ml of 20 mM
Tris-HCl (pH 8.0) containing 25 mM NaCl and 5 mM
-mercaptoethanol
(
-ME), and the resulting solution was then
dialyzed twice against
2 liters of the same buffer. The dialyzed sample
was loaded onto
a Q-Sepharose anion-exchange column (Pharmacia) (2.5 by
14 cm)
which had been equilibrated with the same buffer. TrmE was
eluted
with the same buffer using a gradient of 0.025 to 1 M NaCl.
Fractions
containing TrmE were pooled and precipitated with ammonium
sulfate
at 80%
saturation.
The ammonium sulfate precipitate was solubilized in 10 ml of 10 mM
potassium phosphate buffer (pH 7.0) containing 50 mM NaCl
and 5 mM
-ME, and the solution was dialyzed twice against 2 liters
of the
same buffer. The dialyzed sample was then loaded onto a
hydroxylapatite
column (Bio-Rad) (2.5 by 5 cm) which had been
equilibrated with the
same buffer. TrmE was eluted with the same
buffer using a gradient of
10 to 500 mM potassium phosphate. Fractions
containing TrmE were pooled
and precipitated with ammonium sulfate
at 80% saturation. The
precipitate was solubilized in 2 ml of
20 mM Tris-HCl (pH 7.5)
containing 50 mM NaCl, 5 mM
-ME, and
10% glycerol, and the solution
was dialyzed twice against 1 liter
of the same buffer. The dialyzed
sample was then loaded onto a
Blue column (Pharmacia) (1.0 by 30 cm)
which had been equilibrated
with the same buffer. TrmE was eluted with
the same buffer using
a gradient of 0.05 to 1.5 M NaCl. Fractions
containing TrmE were
pooled and precipitated with ammonium sulfate at
80% saturation.
The precipitate was solubilized in 2 ml of 20 mM
Tris-HCl (pH
7.5) containing 50 mM NaCl, 5 mM
-ME, and 10%
glycerol, and the
solution was dialyzed twice against 1 liter of the
same buffer.
TrmE protein thus obtained was approximately 95% pure as
judged
by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis analysis
(Fig.
2A).
Typically, 20 mg of purified TrmE was obtained from
a 3-liter culture.
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FIG. 2.
Purification, oligomer formation, and GTPase
activity of TrmE. (A) Purified TrmE was analyzed by sodium dodecyl
sulfate-12.5% polyacrylamide gel electrophoresis, and the gel was
stained with Coomassie brilliant blue. Lane 1, bovine serum albumin (66 kDa) and trypsin inhibitor (21.7 kDa) as molecular mass standards; lane
2, purified TrmE (2 µg). (B) Purified TrmE was applied to a Superdex
G200 (Pharmacia) column which had been equilibrated with 20 mM
KPO4 buffer (pH 8.0) containing 50 mM NaCl. Thyroglobulin
(669 kDa), apoferritin (443 kDa), -amylase (200 kDa), alcohol
dehydrogenase (150 kDa), bovine serum albumin (66 kDa), ovalbumin
(43 kDa), and carbonic anhydrase (29 kDa) were used as molecular mass
standards. Peaks at 260, 160, and 52 kDa are designated a, b, and c,
respectively, as shown. (C) Hydrolytic activity from GTP to GDP of
TrmE. The GTPase assay was carried out in a 50-µl reaction
mixture of 50 mM Tris-HCl (pH 9.0) containing 200 mM KCl, 5 mM
MgCl2, 1 mM DTT, 10 µM [ -32P]GTP, and 10 µg of purified TrmE protein at 70°C for 10 min. The reaction was
terminated by transfer of 5 µl of samples to 10 µl of ice-cold 20 mM EDTA. Portions of the terminated reaction mixture were spotted onto
a polyethyleneimine-cellulose thin-layer chromatography plate, which
was developed in 0.75 M KH2PO4 (pH 3.65). The
plate was autoradiographed to identify hydrolyzed products of GTP.
Spots corresponding to GTP, GDP, and GMP (lane 5) were identified by UV
shadowing. Lanes 1 and 2, incubations without TrmE for 0 and 60 min,
respectively; lanes 3 and 4, incubations with 10 µg of TrmE for 0 and
60 min, respectively.
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Interestingly, when the purified
T. maritima TrmE (50,650 Da) was applied to a gel filtration column, it was eluted into three
peaks (Fig.
2B): peak a at 260 kDa, peak b at 160 kDa, and peak
c at 52 kDa. This indicates that TrmE mostly exists as oligomers
in
solution: primarily as a trimer (peak b) and a hexamer (peak
a), with a
minor fraction as a monomer (peak c).
E. coli TrmE
(49,200 Da) is also known to be eluted from a gel filtration column
in
a range of 50 to 250 kDa (
6). Thus, oligomerization might
be
a common characteristic of the TrmE subfamily, and it is possible
that at least one of the four well-conserved regions (regions
I to IV)
is involved in the
oligomerization.
GTPase activity of TrmE.
In order to demonstrate that the
purified T. maritima TrmE protein is indeed a GTP-binding
protein and possesses a GTPase activity, purified TrmE was
incubated for 60 min at 70°C with [-32P]GTP and the
reaction mixture was analyzed by thin-layer chromatography. As shown in
Fig. 2C, GTP was converted to GDP by TrmE, indicating that T. maritima TrmE has a GTPase activity.
Next, we determined the optimum conditions for GTPase activity. GTP
hydrolysis occurred linearly with up to 30 µg of protein
per reaction
mixture (data not shown). With 10 µg of protein,
GTP hydrolysis
occurred linearly for up to 60 min of incubation
(data not shown). To
determine the optimal reaction temperature,
GTP hydrolysis assays were
carried out at 30, 40, 50, 60, 70,
80, and 90°C. As shown in Fig.
3A, maximum activity was found
at 80°C.
However, the higher temperature caused a higher background:
0.7 and
1.9% of input GTP was hydrolyzed without protein in 10
min at 80 and
90°C, respectively, while less than 0.3% was hydrolyzed
at other
temperatures. Therefore, all of the reactions described
below were
carried out at 70°C.
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FIG. 3.
Effects of reaction temperature, pH, and salt
concentration on the GTPase activity of TrmE. (A) The GTPase
assay was carried out in a 50-µl reaction mixture of 50 mM Tris-HCl
(pH 7.5) containing 5 mM MgCl2, 1 mM DTT, 10 µM
[ -32P]GTP, and 10 µg of TrmE for 10 min at different
temperatures. The GTPase assay reaction was stopped by adding
activated charcoal followed by centrifugation, and the release of
32Pi in the supernatant was assayed using a
liquid scintillation counter. (B) The GTPase assay was carried out
in a 50-µl reaction mixture. Between pH 6.5 and 9.0, the mixture
contained 50 mM Tris-HCl; at pH 9.5 and 10, the mixture contained 50 mM
3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid. Both buffers
contained 5 mM MgCl2, 1 mM DTT, 10 µM
[-32P]GTP, and 10 µg of TrmE, and the reaction was
carried out for 10 min at 70°C. (C) The GTPase assay was carried
out in a 50-µl reaction mixture of 50 mM Tris-HCl (pH 7.5) containing
5 mM MgCl2, 1 mM DTT, 10 µM [-32P]GTP,
10 µg of TrmE, and different concentrations (0 to 500 mM) of KCl or
NaCl for 10 min at 70°C. The reaction was carried out at least twice,
and the average value for each point was used. Background values
(without protein) were subtracted.
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The effects of pH, salt, and Mg
2+ on GTPase activity
were subsequently examined. TrmE was found to prefer alkaline
conditions,
with the optimum activity at pH 9.0, while it had almost no
activity
at neutral and acidic pHs (Fig.
3B). The TrmE GTPase was
found
to be specifically activated by KCl and, as shown in Fig.
3C,
at
a sevenfold-higher rate at 200 mM KCl than at 200 mM NaCl.
At present,
we do not know whether these in vitro characteristics
of pH and
K
+ concentration reflect in vivo conditions. The
Mg
2+ ion is also required for GTPase activity, which
reached its highest
level at greater than 1 mM Mg
2+ (data
not
shown).
Thus, the optimum conditions for TrmE GTPase activity were
determined to be as follows: the reaction is carried out at 70°C
for
10 min in 50 mM Tris-HCl (pH 9.0) containing 200 mM KCl, 5
mM
MgCl
2, 1 mM dithiothreitol (DTT), and 10 µM GTP with 10 µg
of TrmE in a 50-µl reaction mixture. Biochemical parameters of
the TrmE GTPase activity were determined under optimum conditions.
Using GTP concentrations from 0.001 to 3 mM, the Lineweaver-Burk
plot
was determined (Fig.
4).
Km and
Vmax for the
GTPase activity
of TrmE were estimated to be 833 µM and 37 µM/min, respectively.
kcat was calculated to
be 9.3 min
1. This indicates that
T. maritima
TrmE has a very high intrinsic
GTP hydrolysis rate. Note that the
concentration of GTP in exponentially
growing bacterial cells is about
1 mM (
20).
E. coli TrmE also
shows a very high
intrinsic GTP hydrolysis rate (
Km, 378 µM;
kcat,
26 min
1) (
6),
which is comparable to
T. maritima TrmE, suggesting
that the
very high intrinsic GTP hydrolysis rate might be a common
characteristic among TrmE proteins from various species. It would
be
interesting to know if the hydrolysis rate is modulated by
an effector
molecule in the cells.
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FIG. 4.
Lineweaver-Burk plot of TrmE GTPase activity. The
GTPase assay was carried out in a 50-µl reaction mixture of 50 mM
Tris-HCl (pH 9.0) containing 200 mM KCl, 5 mM MgCl2, 1 mM
DTT, 10 µg of TrmE, and different concentrations (0.001 to 3 mM) of
GTP for 10 min at 70°C. Each point is the average of at least two
experiments. Background values (without protein) were subtracted. Vo,
initial rate of reaction.
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To determine the substrate specificity, competition experiments were
carried out with various nucleotides added at a 300-fold
excess over
the GTP used in the reaction. As shown in Fig.
5,
only GTP, GDP, and dGTP were found to
compete GTP hydrolysis.
The high substrate specificity of TrmE to
guanine nucleotides
was further confirmed by the fact that TrmE has no
detectable
ATPase activity under the conditions used for the GTPase
assay
(data not shown). Note that GTP binding of
E. coli
TrmE was shown
to be competed with GTP, GDP, and dGTP (
6).
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FIG. 5.
GTPase assay in the presence of competitors. The
GTPase assay was carried out in a 50-µl reaction mixture of 50 mM
Tris-HCl (pH 9.0) containing 200 mM KCl, 5 mM MgCl2, 1 mM
DTT, 10 µM [-32P]GTP, and 10 µg of TrmE for 10 min
at 70°C in the presence of each competitor at a final concentration
of 3 mM. Each point is the average of at least two independent
experiments. Competitors used are shown, and activities are relative to
the value with no competitor (100%).
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Conclusions.
We have demonstrated that T. maritima
TrmE possesses a GTPase activity having a very high intrinsic GTP
hydrolysis rate. Although its Km value is quite
high (833 µM) in comparison with the Km value
of E. coli Era (10 µM) (7), the TrmE GTPase
activity is likely to be significantly regulated by its effector
molecule(s) in the cells. The identification of such an effector
molecule is important for our understanding of TrmE function, which is known to be essential for cell growth in E. coli
(6).
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ACKNOWLEDGMENTS |
K. Yamanaka and J. Hwang contributed equally to this work.
We are very grateful to F. E. Jenney, Jr., for providing genomic
DNA of T. maritima.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Ln.,
Piscataway, NJ 08854. Phone: (732) 235-4115. Fax: (732) 235-4559. E-mail: inouye{at}umdnj.edu.
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0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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