From the Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90095-1569
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ABSTRACT |
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We have identified a new type of
S-adenosyl-L-methionine-dependent
methyltransferase in the cytosol of Escherichia coli that is expressed in early stationary phase under the control of the RpoS
Our laboratory has been interested in the protein
L-isoaspartate (D-aspartate)
O-methyltransferase (EC 2.1.1.77), an enzyme that catalyzes
the methyl esterification of spontaneously altered residues in a
pathway that can lead to the conversion of isomerized aspartyl residues
to normal aspartyl residues in a net repair reaction (1). We have
postulated that the physiological role of this methyltransferase is to
preserve the integrity of the polypeptide chain in the face of
age-dependent non-enzymatic reactions that lead to
alterations in its configuration (2, 3). In the bacterium
Escherichia coli, this enzyme is required for optimal survival of stationary phase cells against environmental stresses (4,
5), presumably functioning to maintain proteins in active configurations under conditions where protein synthesis to replace damaged proteins is limited.
In the course of our studies of the protein L-isoaspartate
methyltransferase encoded by the pcm gene in E. coli, we found an activity in cytosolic extracts that appeared to
catalyze methyl ester formation but was not dependent upon the
pcm gene product. We have now traced this activity to that
of a previously undescribed small molecule methyltransferase that is
active on trans-aconitate, an apparently non-enzymatically
formed derivative of the citric acid cycle intermediate
cisaconitate. Since both types of methyltransferase activities are directed to substrates that can be formed by spontaneous age-related processes, we were interested in characterizing the trans-aconitate methyltransferase. We found that the
expression of this activity is dependent on the stationary phase
specific Preparation of E. coli Cytosol for Methyltransferase Assay
E. coli strains and plasmids used in this study are
described in Table I. For analytical
studies, E. coli cells were grown to stationary phase (20 h)
in 5 ml of Luria-Bertani (LB) broth or M9 medium containing
D-glucose supplemented with thiamine (18 µg/ml) and
leucine (40 µg/ml) (Ref. 10, section A.3). When appropriate, 100 µg/ml ampicillin, 50 µg/ml kanamycin, 20 µg/ml chloramphenicol, or 20 µg/ml tetracycline were added. Cells were collected by
centrifugation at 5,000 × g at 4 °C for 10 min. The
cell pellet was resuspended in 0.5 ml of buffer containing 5 mM disodium EDTA, 10% glycerol, 25 µM
phenylmethylsulfonyl fluoride in 5 mM potassium phosphate buffer at a final pH of 7.0. Cells were lysed by sonication in an ice
bath using the microtip of a Branson model W350 instrument at an output
control setting of 4 for three sets of 5 pulses separated by 30-s
cooling pauses. The extract was centrifuged at 12,000 × g at 4 °C for 10 min, and the supernatant was used as a
cytosolic fraction. The protein concentration was determined by the
method of Lowry et al. (11) after 10% trichloroacetic acid
precipitation of the samples.
factor. This enzyme catalyzes the monomethyl esterification of
trans-aconitate at high affinity (Km = 0.32 mM) and cis-aconitate, isocitrate, and
citrate at lower velocities and affinities. We have purified the enzyme
to homogeneity by gel-filtration, anion-exchange, and hydrophobic
chromatography. The N-terminal amino acid sequence was found to match
that expected for the o252 open reading frame at 34.57 min
on the E. coli genomic sequence whose deduced amino acid
sequence contains the signature sequence motifs of the major class of
S-adenosyl-L-methionine-dependent methyltransferases. Overexpression of the o252 gene
resulted in an overexpression of the methyltransferase activity, and we
have now designated it tam for
trans-aconitate
methyltransferase. We have generated a knock-out strain of
E. coli lacking this activity, and we find that its growth
and stationary phase survival are similar to that of the parent strain.
We demonstrate the endogenous formation of trans-aconitate
methyl ester in extracts of wild type but not
tam
mutant cells indicating that
trans-aconitate is present in E. coli. Since
trans-aconitate does not appear to be a metabolic intermediate in these cells but forms spontaneously from the key citric
acid cycle intermediate cis-aconitate, we suggest that its
methylation may limit its potential interference in normal metabolic
pathways. We have detected trans-aconitate
methyltransferase activity in extracts of the yeast Saccharomyces
cerevisiae, whereas no activity has been found in extracts of
Caenorhabditis elegans or mouse brain.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
factor RpoS. We have purified and characterized this
enzyme, identified its gene, and characterized a knock-out strain
lacking this activity. We were able to show that
trans-aconitate is an endogenous substrate for the enzyme
and that the product is its monomethyl ester.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Strains and plasmids used
trans-Aconitate Methyltransferase Assay
Enzyme activity was measured using a modification of the protein carboxyl methyltransferase assay (7). Unless otherwise stated, the assay mixture consisted of 2 µl of 20 mM trans-aconitic acid (Sigma) in 0.4 M sodium HEPES, pH 7.5, 5-15 µl of the enzyme preparation, 5 µl of 80 µM S-adenosyl-L-[methyl-14C]methionine ([14C]AdoMet1; specific radioactivity about 110 cpm/pmol, 53 mCi/mmol, Amersham Pharmacia Biotech), 10 µl 0.4 M sodium HEPES, pH 7.5, and water to a total volume of 40 µl. Samples were incubated at 37 °C for 5-30 min, and the reactions were quenched by adding 40 µl of freshly prepared 2 M NaOH. Sixty µl of this mixture was immediately spotted onto an accordion-pleated 1.5 × 8-cm piece of thick filter paper (Bio-Rad 165-0962), and the paper was placed in the neck of a 20-ml vial containing 5 ml of Safety-Solve scintillation fluid (Research Products International Corp.), capped, and incubated for 2 h at room temperature. Radioactivity was determined by liquid scintillation counting in a Beckman LS6500 counter after removal of the filter paper.
In initial experiments, citrate was used as a methyl-accepting substrate. Here, the reaction contained 20 µl of 0.2 M sodium citrate, pH 6.0 (Fisher, ACS-certified), 15 µl of enzyme preparation, and 5 µl of 80 µM [14C]AdoMet as described above. Samples were incubated at 37 °C for 20 min and analyzed as described above. Other substrates tested included cis-aconitic acid, DL-isocitrate (threo-DsLs-isocitrate; trisodium salt, 93-98%), (2R,3S)-isocitrate (threo-Ds(+)isocitrate; monopotassium salt, approximately 99%), fumaric acid, tricarballylic acid, and malic acid, all obtained from Sigma, and succinic acid and oxalacetic acid obtained from Fisher.
Purification of the E. coli trans-Aconitate Methyltransferase
Preparation of E. coli Cytosol-- Four flasks each containing 2 liters of LB media were each inoculated with 2 ml of an overnight culture of E. coli strain MC1000 and were grown to stationary phase at 37 °C for 20 h with shaking. Cells were collected by centrifugation at 5,000 × g at 23 °C for 15 min. The cell pellet (29.1 g) was washed three times with 400 ml of buffer A (50 mM Tris-HCl, 5 mM disodium EDTA, 300 mM NaCl, pH 8.0) and then resuspended in 50 ml of buffer B (50 mM Tris-HCl, 5 mM disodium EDTA, 25 µM phenylmethylsulfonyl fluoride, pH 8.0) at 4 °C. Cells were disrupted by passing them twice through a French press cell (SLM Aminco) at 20,000 pounds/square inch. The cytosolic fraction was obtained by centrifugation at 23,000 × g for 30 min at 4 °C. This supernatant was then further centrifuged at 100,000 × g for 60 min at 4 °C to remove any residual membrane material.
Ammonium Sulfate Precipitation-- An equal volume of 90% saturated ammonium sulfate (4 °C) was gradually added to the cytosol with stirring, followed by additional stirring at 4 °C for 30 min. The mixture was then centrifuged at 23,000 × g at 4 °C for 30 min. The protein pellet was redissolved in 20 ml of buffer B to a protein concentration of approximately 28 mg/ml.
Superdex S-200 Chromatography-- An aliquot (5 ml) of the redissolved ammonium sulfate pellet was loaded onto a Superdex-200 (Amersham Pharmacia Biotech) gel filtration column (1.5 cm in diameter × 58 cm in height, 102-ml bed volume), pre-equilibrated at 4 °C with buffer C (50 mM Tris-HCl, 5 mM disodium EDTA, pH 8.0). The column was eluted at 18 ml/h, and fractions of 1.2-ml were collected. The activity was eluted at fractions 49-58, and these fractions were pooled and stored at 4 °C. The material from 4 column runs was combined to use in the following step.
DEAE-cellulose Anion-exchange Chromatography-- The active pool from the Superdex-200 column (total volume of 48 ml) was loaded onto a DE52 anion-exchange column (Whatman; 2 cm in diameter × 12.7 cm in height, 40-ml bed volume) pre-equilibrated at 4 °C with buffer C. After sample loading, the column was washed with 3 column volumes of the equilibration buffer. The enzyme was then eluted with a linear sodium chloride gradient (0-0.8 M in the equilibration buffer, total of 250 ml) followed by a 5 column volumes of a high salt wash (1.0 M sodium chloride in the equilibration buffer) at 4 °C. The flow rate was 19.5 ml/h, and 2.8-ml fractions were collected. Activity was found to elute between sodium chloride concentrations of 300 and 400 mM between fractions 102 and 112. These active fractions were pooled and stored at 4 °C.
Hydrophobic Interaction Chromatography-- Potassium monobasic phosphate was dissolved in concentrated buffer C, the pH was adjusted to 8.1 with KOH, and the solution was diluted to give a final concentration of 1 M phosphate in buffer C. This solution was added to the active pool from the DE52 anion-exchange column to bring the potassium phosphate concentration to 0.6 M. This material was then applied to a phenyl-Sepharose column (Amersham Pharmacia Biotech; 1 cm diameter × 10 cm height, 7.8-ml bed volume) pre-equilibrated with 0.6 M potassium phosphate in buffer C. The flow rate was 18 ml/h, and 2.5-ml fractions were collected. After loading the sample, the column was washed with 5 column volumes of the equilibration buffer and then eluted with a linear gradient of potassium phosphate (0.6-0 M in the equilibration buffer). Enzyme activity was found to elute between 0 and 0.1 M potassium phosphate concentrations.
Amino Acid Sequencing
N-terminal amino acid sequence analysis was performed by Dr. Audree Fowler at the UCLA Protein Microsequencing Facility with a Porton 2090E gas-phase sequencer with on-line HPLC detection. The active pool from the DE52 column (fractions 103-110, total volume of 19.6 ml) was added to an equal volume of 25% (w/w) trichloroacetic acid, mixed well by vortexing, and incubated at 4 °C while rotating slowly overnight. The mixture was centrifuged at 15,000 × g for 30 min at 4 °C, and the protein pellet was then dissolved in 100 µl of 1× sample buffer for SDS gel electrophoresis (Ref. 10, section 18.47-18.55). After polyacrylamide gel electrophoresis, the separated polypeptides were electroblotted onto a polyvinylidene difluoride membrane in 25 mM Tris base, 10 mM glycine, 0.5 mM dithiothreitol, in 10% methanol, 90% water (v/v) at pH 9. This membrane was then stained with Coomassie Brilliant Blue R250, and the polypeptide band at 29 kDa corresponding to the trans-aconitate methyltransferase was excised and subjected to automated Edman sequencing.
Cloning and Disruption of the trans-Aconitate Methyltransferase Gene in E. coli
A 3.1-kb DNA fragment containing the entire
trans-aconitate methyltransferase gene (o252) and
flanking regions was amplified by polymerase chain reaction (PCR) from
template DNA in MC1000 cells (12) using Taq polymerase
(Promega) at 2.5 mM magnesium chloride and an annealing
temperature of 60 °C. The primers were KO-5
(5'-TATGACTACGAAGCGGATCCTAATGGCA, corresponding to bases 1362 to
1335 from the translation start site of o252
with the underlined nucleotides changed to create a BamHI
site) and KO-3 (5'-GCGTATTGAGAATGGGATCCTAATCACG
corresponding to the reverse complement of bases 1714-1741 with the
underlined nucleotides changed to prevent hairpin formation and to
create another BamHI site). This fragment was purified by
gel electrophoresis using Geneclean II (Bio 101), cut with
BamHI, and then ligated into the BamHI site
within the multicloning site of the pUC19 vector to generate the
plasmid pHC108 (Table I). This plasmid, pHC108, was then used to
construct a null mutation in the o252 gene by blunt-end
ligation of a 1.5-kb chloramphenicol resistance
(Cmr) cassette (13) at the unique
AgeI site within the gene to create pHC109. The chromosomal
o252 gene was then replaced with the
Cmr disrupted gene in pHC109 by homologous
recombination in strain JC7623, which does not support plasmid
replication (14). pHC109 was transformed into
CaCl2-competent JC7623 cells, and Cmr colonies
were selected on a plate containing 20 µg/ml chloramphenicol. The
loss of the vector in the recipient strain was confirmed by screening
for ampicillin sensitivity. This disrupted o252 gene was
subsequently transduced into the MC1000 background by P1 transduction (15). The disruption of the o252 gene was confirmed by the
PCR amplification of a 3.96-kb product using a primer
(5'-GATTCAGTACGCCAAATGTG) corresponding to genomic sequence upstream of
the KO-5 primer described above and a primer OE-3 (described below)
corresponding to a sequence downstream of the stop codon for
o252 gene. We also confirmed the disruption by the detection
of the expected 3.44-kb EcoRV, 6.35-kb HindIII,
and 0.60- and 1.35-kb EcoRI fragments using a random-primed
probe corresponding to the Cmr gene in Southern
blot hybridization (data not shown).
Overexpression of trans-Aconitate Methyltransferase
The trans-aconitate methyltransferase gene was
PCR-amplified from colonies of E. coli strain MC1000 as
described above using the primers OE-5 (5'-
CGGGAGTAAACATATGTCTGACTGG; corresponding to
bases 13 to +12 from the translation start site with the underlined bases changed to create a 5' NdeI site) and OE-3
(5'-ACCACTGGATCCCATATGCAACGC; corresponding to
the reverse complement of bases +848 to +871 with the underlined based
changed to create a BamHI site and to prevent hairpin
formation; the stop codon is located at bases +757 to +759). The
884-base pair PCR fragment was cleaved with NdeI and
BamHI and the large fragment purified as described above. This fragment was then cloned into the corresponding sites in the
multicloning site of the pT7-7 vector (16) to generate pHC107. DNA
sequence analysis using both oligonucleotides described above as
primers showed that no mutations were introduced during the cloning
procedure. The plasmid was then transferred into BL21(DE3) cells
(Invitrogen) for expression. An aliquot of an overnight culture of the
transformed cells (20 µl) was diluted into 20 ml of fresh LB medium,
incubated with shaking at 37 °C, and cultured to an
A600 nm of 1.0. Isopropyl-
-D-thiogalactopyranoside was added to a final
concentration of 1 mM to the culture, and the cells were
incubated with shaking for another 2 h. Cytosolic fractions were
then prepared and assayed for trans-aconitate
methyltransferase activity as described above. The enzymatic activity
of this preparation was 100-150 nmol/min/ml (specific activity 12-22
nmol/min/mg protein), and it was used as a concentrated source of
enzyme in the enzyme kinetic assays and in the experiments to
characterize the methyl acceptors. A control extract was prepared where
the same strain was grown in the absence of plasmid. The specific
activity of this extract was 0.020 nmol/min/mg protein, indicating that
the degree of overexpression was about 630-fold.
High Performance Liquid Chromatography
Analysis of the substrates and products was carried out with a Waters HPLC system. An Alltech Partisil SAX anion-exchange column (250 mm length × 4.6 mm inner diameter; 10-µm resin bead diameter) was used for initial separations. The column was equilibrated with 50 mM potassium phosphate, pH 4.5, and eluted at 1 ml/min at room temperature. After each run, the column was regenerated with 500 mM potassium phosphate, pH 4.5, prior to re-equilibration with the starting buffer. For reverse-phase separations, an Alltech Econosphere C18 column (250 mm length × 4.6 mm inner diameter; 5-µm spherical resin bead diameter) was used in a two-solvent system. Solvent A is 0.1% trifluoroacetic acid in water, and solvent B is 0.1% trifluoroacetic acid, 99.5% acetonitrile, and 0.4% water. The column was eluted at room temperature at a flow rate of 1 ml/min for 20 min in solvent A, followed by a linear gradient over 20 min from 100% solvent A to 100% solvent B, followed by 10 min of 100% solvent B. The column was re-equilibrated with 100% buffer A. In each case, 1-min fractions were collected.
The UV profile was monitored by Waters model 441 absorbance detector at 214 nm, and chromatographs were recorded with the PowerChrom system from ADInstruments. Radiolabeled products were detected by counting 100 µl of each in 5 ml of scintillation fluid.
Thin Layer Chromatography
Thin layer chromatography was also used to analyze the products of the methylation reaction after the method of Otten and Mehltretter (17). Polyester-based 60-Å silica gel-coated plates were used (Whatman PE SIL G, 250 µm layer). The solvent is benzene/methanol/acetic acid (45:16:4, v/v/v) and is prepared fresh daily and used to pre-equilibrate the chamber for 2 h before each run at room temperature. Samples (1-5 µl) were spotted in each lane in 1-µl aliquots, and each spot was allowed to air-dry before further application of sample or chromatography. The solvent front was allowed to migrate approximately 18 cm on the plate, then its position was marked and the plate was air-dried in a hood, followed by baking at 105 °C in a vacuum oven for 1 h. Carboxylic acid-containing compounds were detected as yellow spots on a blue background after spraying the plate with 0.04% bromcresol green dissolved in 95% ethanol, pH 8.0.
Synthesis and Characterization of trans-Aconitate Methyl Esters
Chemical Methylation-- trans-Aconitic acid (4.8 mg) was incubated with 60 µl of methanol and 1 µl of concentrated (12 M) HCl for 16 h at room temperature. The sample was vacuum-dried in Speedvac apparatus and dissolved in 138 µl of H2O to give a final concentration of trans-aconitate derivatives of 0.2 M. An aliquot of the sample (10 µl) was chromatographed on the SAX HPLC anion-exchange column as described above. The peaks were collected, and an aliquot of each peak was re-chromatographed on the C18 reverse-phase column as described.
Enzymatic Methylation-- The reaction mixture contains 5 µl of 0.02 M trans-aconitate in 0.4 M sodium HEPES, pH 7.5, 10 µl of 0.4 M sodium HEPES, pH 7.5, 1 µl of a preparation of cytosol from BL21 cells overexpressing the trans-aconitate methyltransferase (7.72 µg protein, 12.6 pmol/min/µg protein), 17.5 µl of 8.16 mM S-adenosyl-L-methionine (AdoMet) in water, and 2.5 µl [14C]AdoMet to a total volume of 42.5 µl made up by H2O. The reaction was carried out at 37 °C for 24 h. Forty µl of the sample was purified as described above. The methylated trans-aconitate was followed by radioactivity.
Mass Spectroscopy--
Mass spectroscopy was performed by Dr.
Kym Faull at the UCLA Mass Spectrometry Facility. HPLC fractions from
the C18 reverse-phase column were collected and dried in a SpeedVac.
The dried HPLC samples were redissolved in 20 µl of
water/acetonitrile/triethylamine (50:50:0.1, v/v/v), and aliquots were
injected into an electrospray ionization source attached to a
quadrupole mass spectrometer (Perkin-Elmer, Thornhill, Canada, API III;
3.5 Kv ion spray voltage, spray nebulization with
hydrocarbon-depleted air ("zero" grade air, 40 pounds/square inch,
0.6 liters/min; Zero Air Generator, Peak Scientific, Chicago, IL),
curtain gas (0.6 liters/min) from the vapors of liquid nitrogen; mass
resolution set so the isotopes of the polypropylene
glycol/NH4+ singly charged ion at
m/z 906 were resolved with 40% valley) scanning from
m/z 120-250 in the negative ion mode. Spectra were collected (step size 0.3 Da, dwell time 20 ms/step, 6.7 s/scan, orifice
at
60 V), and the resulting spectra were summed then background
subtracted with software supplied with the instrument.
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RESULTS |
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Identification of a Novel Methyltransferase Activity in E. coli--
In the course of studies quantitating protein
L-isoaspartate O-methyltransferase activity
in various strains, we measured methyl esterification in cytosolic
extracts in the presence and absence of added
L-isoaspartyl-containing methyl-accepting peptide KASA(isoD)LAKY. [14C]Methyl esters formed when extracts
are incubated with [14C]AdoMet in a sodium citrate buffer
were hydrolyzed in base to generate volatile
[14C]methanol that can be separated from unreacted
[14C]AdoMet and other non-volatile species and
quantitated. Although we found that the L-isoaspartyl
peptide-dependent activity correlated well with the
presence of the pcm gene for the isoaspartyl
methyltransferase, we were surprised to find that the "endogenous"
activity in the absence of added peptide was much higher in extracts
from strains with an intact rpoS gene than in extracts from
strains mutated in this gene (Table II).
The rpoS gene is located about a kilobase downstream from
the pcm gene and codes for a specific factor that is
required for the expression of a number of genes in stationary phase
cells (for a review, see Ref. 18). This result suggested that either
rpoS or one of the genes it regulates might also have a
methyl esterification activity.
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We then began to investigate the nature of the RpoS-dependent endogenous methyl esterification activity using extracts of the strain HC1011 that lacks the pcm gene so there would be no contribution of the L-isoaspartyl methyltransferase to the methylation activity. We found that about 78% of the total activity was localized in the cytosolic fraction, and 22% of the activity was found in the membrane pellet fraction. The combination of membrane and cytosolic fractions did not increase the activity over that of cytosolic fractions alone (data not shown). The production of the endogenous methylated product from cytosolic extracts was found to be linear with time and with the amount of cytosolic extract used (data not shown). The activity appeared to be a protein because treatment of the extract with proteinase K resulted in a complete loss of activity and because heating the extract greatly reduced the activity. Finally, we showed that the activity could be inhibited by S-adenosyl-L-homocysteine, a product of the reaction and an effective inhibitor of most AdoMet-dependent methyltransferases (19). We found 72% inhibition with 0.38 mM S-adenosyl-L-homocysteine and essentially complete inhibition at 0.94 mM (data not shown).
Initial Characterization of Substrates and Products-- The formation of volatile radioactivity by the RpoS-dependent methyltransferase in the assay described above was dependent upon hydrolysis of the reaction products, suggesting that a methyl ester linkage was formed. No radioactivity was detected when water replaced the 2 M NaOH quenching solution. There was no increase in activity when 7 M NaOH was used as a quench; 74% of the activity was detected with 1 M NaOH and only 49% activity found with 0.2 M NaOH. Only 2, 3, and 8% of the maximum activity was found when 1, 2, and 7 M HCl was used as a quench (data not shown).
No loss of activity was seen after dialysis of the extract, initially suggesting that a macromolecule may be the methyl acceptor. However, we found that the endogenous activity was dependent upon the presence of the citrate buffer in the assay mixture. When the citrate was replaced by Tris, phosphate, acetate, or MES buffers of similar pH, no activity was seen (data not shown). The role of citrate in the reaction did not appear to be that of a metal chelator because there was no activity in a buffer containing 5 mM sodium EDTA. These results suggested that citrate could itself be the substrate for the reaction where one or more of its three carboxyl groups could be methyl-esterified. To ask whether compounds structurally related to citrate would be better or worse substrates, we assayed extracts with a variety of tricarboxylates and dicarboxylates. While we found that succinate, fumarate, malate, oxalacetate, and tricarballylate gave no activity when substituted for citrate, even better activity was found with cis-aconitate, DL-isocitrate, (2R,3S)-isocitrate, and trans-aconitate. From the concentration dependence of the activity, we estimated an apparent Km value for trans-aconitate of about 0.3 mM, a value at least 16-fold lower than that measured for citrate (7.1 mM), cis-aconitate (33 mM), (2R,3S)-isocitrate (9.1 mM), or DL-isocitrate (5 mM). We found that the reaction with citrate had a maximal velocity of only 7.5% that of trans-aconitate, whereas the reaction with cis-aconitate, (2R,3S)-isocitrate, DL-isocitrate had comparable maximal velocities (108, 49, and 78%) (data not shown). By using trans-aconitate as a substrate, we found that the apparent Km for [14C]AdoMet is about 5 µM, whereas the pH dependence of the enzyme activity demonstrated a broad maximum from pH 6 to 8 decreasing at higher or lower pH values with half-maximal activities at about pH 5.5 and pH 8.5 (data not shown).
trans-Aconitate-dependent Methyltransferase Activity
Rises as Cells Enter Stationary Phase and Then Decreases--
Because
the methyltransferase activity required the presence of the RpoS
stationary phase factor (Table II) whose concentration increases
near the end of exponential growth phase (20), we assayed this enzyme
during various stages of cell growth using trans-aconitate
as a substrate. We found that the specific activity of the
methyltransferase increases dramatically in late exponential phase and
peaks at the transition into stationary phase (Fig. 1A), paralleling the known
accumulation of RpoS (21). The specific activity of the
methyltransferase then decreases dramatically after 24 h in
stationary phase (Fig. 1B), which also correlates with the
decrease in the concentration of RpoS (21). There is little or no
detectable activity after 72 h in stationary phase. We also
observed that the specific activity of
trans-aconitate-dependent methyltransferase is
3-4-fold higher in the cytosolic fractions obtained from cells
cultured in rich LB media than from cell cultured in minimal (M9) media
with glucose (data not shown). This difference may also reflect the
higher level of RpoS in rich media compared with minimal media (20,
21).
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Purification of trans-Aconitate Methyltransferase--
From the
results presented above, it is possible that either RpoS has a
methyltransferase activity or that it is essential for the
transcription of the methyltransferase gene. To clarify this issue, the
methyltransferase was purified by ammonium sulfate precipitation, gel
filtration chromatography on Superdex 200, anion-exchange
chromatography on DE52 resin, and hydrophobic chromatography on
phenyl-Sepharose resin as described under "Experimental
Procedures." A single peak of activity was found in each
chromatographic step suggesting that isozymes are not present (Fig.
2). Characterization of the polypeptide
composition of each fraction by polyacrylamide gel electrophoresis in
sodium dodecyl sulfate (SDS) revealed a polypeptide of about 29 kDa
that is progressively enriched during the purification (Fig.
3). In the final step of hydrophobic
chromatography, only a single band at 29 kDa was observed whose
concentration corresponded directly to the methyltransferase activity
(data not shown). The overall purification of 594-fold (Table
III) suggested that the enzyme made up
approximately 0.2% of the total cytosolic protein in early stationary
phase. Additional native gel filtration experiments showed that the
methyltransferase eluted between the molecular weight markers ovalbumin
(43 kDa) and the human L-isoaspartyl methyltransferase (25 kDa) and thus appears to be composed of a single polypeptide chain.
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Identification of the E. coli Gene Encoding the trans-Aconitate
Methyltransferase--
Microsequencing of the purified 29-kDa
polypeptide demonstrated an N-terminal amino acid sequence of
SDXNKQLYLQFMAEMS, where the assignment of residues 1, 2, and
4 was more certain than residues 5-16, and no assignment could be made
for residue 3. This sequence, including an assumed N-terminal
methionine residue, was used to search against the
GenBankTM protein data base using ungapped BLAST (22). The
best match corresponded to the N terminus of the deduced product of the
o252 open reading frame in the newly sequenced E. coli genome (23) where identities were found at 11 of the 17 positions (Fig. 4). This previously
uncharacterized gene, at 34.57 min on the
chromosome,2 potentially
encodes a polypeptide of 28,876.4 Da (lacking the initiator methionine
which would be expected to be removed (25)) that corresponds to the
29-kDa polypeptide purified. The deduced sequence also contains the
four well conserved motifs (I, post-I, II, and III) in a variety of
methyltransferases (Ref. 26; Fig. 4).
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Overexpression of the trans-Aconitate Methyltransferase and Methyl-accepting Substrate Characterization-- The o252 gene was PCR-amplified and cloned into a pT7-7 expression vector as described under "Experimental Procedures." We found that the specific activity of the trans-aconitate methyltransferase was increased 630-fold in extracts of BL21(DE3) cells compared with extracts of this strain lacking the plasmid. This result suggests that the o252 gene does indeed encode the methyltransferase activity, and we have now named it tam for trans-aconitate methyltransferase.
The availability of the overexpressed enzyme allowed us to characterize its substrate specificity. Initial velocity measurements at a variety of substrate concentrations confirmed that trans-aconitate was the best substrate with a Km value of 0.32 mM (Table IV). The catalytic efficiencies (Vmax/Km) of the enzyme for cis-aconitate, (2R,3S)-isocitrate, and DL-isocitrate were less than 3% that of trans-aconitate, whereas the catalytic efficiency for citrate was only about 0.4% that of trans-aconitate (Table IV). The values in Table IV obtained for the overexpressed enzyme are generally similar to those found when the cytosol of wild type cells was used as a source of enzyme (see above). Finally, we found that the Km for AdoMet with trans-aconitate was 4.8 µM, a value also similar to that obtained with the non-overexpressed cytosol.
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To exclude the possibility that minor contaminants in the commercial
preparations of trans-aconitate, cis-aconitate,
(2R,3S)-isocitrate, or citrate could represent
all or part of the methyl-accepting activity observed in the
experiments described above, we fractionated each these compounds by
HPLC high resolution anion-exchange chromatography (Fig.
5). Individual fractions were then
assayed as potential methylaccepting substrates for the preparation
of overexpressed trans-aconitate methyltransferase. When
trans-aconitate was fractionated, we found that the
methyl-accepting activity exactly paralleled the elution of
trans-aconitate (monitored by its UV absorbance), suggesting
that this was in fact the methyl acceptor (Fig. 5A). On the
other hand, when the commercial preparation of cis-aconitate was fractionated, a more complex pattern was seen. Here, UV analysis indicated that a small contaminant (approximately 5%) of
trans-aconitate was present. This material was a major
methyl acceptor, however, as would be expected from the results
described above (Fig. 5B). Significantly, however, there was
still a methyl-accepting peak corresponding to the
cis-aconitate peak. Thus, cis-aconitate appears to also be recognized by the methyltransferase but at even lower affinity than suggested by the data in Table IV. When we fractionated (2R,3S)-isocitrate we found that all of the
methyl-accepting activity co-eluted with the major peak of isocitrate
(Fig. 5C), suggesting that isocitrate is also a
methyl-accepting substrate of this enzyme. Finally, fractionation of
citrate showed that the methyl-accepting activity followed the peak of
citrate (data not shown).
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Analysis of the Methyltransferase Reaction Products in
Vitro--
trans-Aconitate can be methyl-esterified to
produce one trimethyl-, three structurally distinct dimethyl-, and
three structurally distinct monomethyl esters of
trans-aconitate. We first analyzed the products of the
chemical methyl esterification of trans-aconitate by
anion-exchange HPLC at pH 4.5. We found UV-absorbing products eluting
at 5.5, 9.9, 11.5, and 15.6 min and a very small peak of residual
trans-aconitate was found at 50.4 min (Fig.
6A). Mass spectral analyses
demonstrated that the 5.5-min peak contained both mono- and dimethyl
derivatives of trans-aconitate and that the 11.5- and 16-min
peaks contained monomethyl trans-aconitate derivatives. No
trans-aconitate derivatives were detected in the 9.9-min
peak. In a parallel experiment, we then compared the elution position
of the enzymatically generated [14C]methyl esters formed
by the E. coli enzyme and trans-aconitate with
[14C]AdoMet (Fig. 6A). About 27% of the
radioactivity was found in the flow-through fractions at 4-8 min in
the position expected for residual [14C]AdoMet. The
remaining 73% of the radioactivity was found to elute in a distinct
peak at 16 min in an amount expected for the methyl ester product of
the reaction. When the material eluting at 16 min was treated with 2 N NaOH, 95% of the total radioactivity was base-labile,
consistent with a methyl ester product (data not shown). This
radiolabeled peak eluted in the same position as one of the peaks of
the chemically synthesized methyl esters corresponding to a monomethyl
ester (Fig. 6A). To determine the nature of this material,
the peaks of material eluting at 16 min from the chromatographic
separations of the enzymatically and chemically synthesized material
were then mixed and further fractionated on a C18 reverse-phase column
(Fig. 6B). Here, the radioactivity was found to elute in a
single peak at about 11 min that also corresponded to a peak of
absorbance of 214 nm. Analysis of this material by mass spectroscopy
indicated that it has an m/e of 187 consistent with one or
more of the monomethyl esters of trans-aconitate. Since
the analysis of the other UV-absorbing peaks in the experiment shown in
Fig. 6A showed that monomethyl esters of
trans-aconitate are also present in the 5.5- and 11.5-min
peaks, these results suggest that enzymatic reaction may be specific
for a single carboxylic acid group of trans-aconitate. In
additional experiments, we prepared the enzymatic product on a larger
scale with the overexpressed enzyme as described under "Experimental
Procedures" and confirmed these results with mass spectral analysis
of the product separated by anion-exchange and reverse-phase
chromatography.
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Characterization of E. coli Cells Lacking the trans-Aconitate
Methyltransferase--
To confirm further our identification and to
study the physiological function of the methyltransferase, we
constructed an E. coli strain where a chloramphenicol
resistance element was inserted into the middle of the tam
gene as described under "Experimental Procedures." Extracts of this
strain, designated HC1014, demonstrated no methyltransferase activity
using trans-aconitate, cis-aconitate, citrate, or
DL-isocitrate as substrates (data not shown). Because this
gene is expressed under the control of the RpoS factor in
stationary phase (Table II and Fig. 1), we measured the survival rates
of the wild type and mutant strains in stationary phase and under
environmental stresses of heat shock, osmotic stress, ethanol
treatment, and oxidative stress in experiments similar to those
described in Visick et al. (4). We found no detectable differences between the wild type and mutant strains under any of the
conditions tested. We also showed that the growth rates were similar in
LB media and in minimal media containing either D-glucose
or acetate as a carbon source (data not shown). Finally, neither strain
was able to grow in a solid or liquid media containing trans-aconitate as the sole carbon source (27), and we could detect no aconitate isomerase activity in wild type or mutant cells using the method of Watanabe et al. (27).
Endogenous Substrates of the trans-Aconitate Methyltransferase in
E. coli--
To characterize the endogenous substrates of this enzyme,
we incubated cytosolic extracts of the parent MC1000 strain
(tam+) and the mutant HC1014 strain
(tam) with
S-adenosyl-L-[methyl-3H]methionine
([3H]AdoMet) in the absence of any exogenous methyl
acceptors. We analyzed the extracts for radioactivity in compounds
present in the parent but not in the mutant strain lacking the
trans-aconitate methyltransferase. We first ether-extracted
the acidified reaction mixtures and chromatographed the ether-soluble
phase on anion-exchange HPLC (Fig.
7A). We found that a peak of
radioactivity was present in the position of the in vitro
enzymatically formed monomethyl ester of trans-aconitate in
the parent strain but no radioactivity at this position in the mutant
strain. To confirm that the endogenous material from the parent strain
was the methyl ester of trans-aconitate, we pooled the
radioactive peak and subjected the material to reverse-phase HPLC. Here
we found that the material again chromatographed as the methyl ester of
trans-aconitate (Fig. 7B). Finally, we pooled the
reverse-phase radioactive peak and subjected it to thin layer chromatography (Fig. 7C). Once again the radioactivity
co-migrated with the methyl ester of trans-aconitate. These
experiments demonstrate that trans-aconitate is present in
E. coli extracts and is an endogenous substrate of the
methyltransferase.
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Presence of the Activity and Gene in Other Organisms-- We assayed the citrate-, DL-isocitrate-, and trans-aconitate-specific activity in cell extracts of Saccharomyces cerevisiae, Caenorhabditis elegans, and mouse brain. The methyltransferase activity is present in yeast but absent in nematodes and mouse brain (Table V). The specific activity of the enzyme in the yeast extract used was about half that seen in extracts of E. coli in stationary phase. Analysis of the GenBankTM data base revealed a homolog of the E. coli trans-aconitate methyltransferase gene in Mycobacterium tuberculosis but, surprisingly, not one in the complete genome of S. cerevisiae. However, examination of several unfinished microbial genomes available through the National Center for Biotechnology Information indicated that there are apparent homologs of trans-aconitate methyltransferase in Deinococcus radiodurans, Pseudomonas aeruginosa, and Yersinia pestis (Fig. 4).
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DISCUSSION |
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We have identified a novel O-methyltransferase in E. coli. It methylates one of the three carboxyl groups of trans-aconitic acid to form a monomethyl ester. The enzyme also recognizes cis-aconitate, (2R,3S)-isocitrate, and citrate but with much higher Km values and/or much lower Vmax values. No reaction is seen with other related metabolites such as succinate, fumarate, malate, or oxalacetate. The fact that it is not active on tricarballylate (the saturated form of aconitate) suggests the importance of an olefinic or hydroxyl function in the recognition of the substrate for the methyl transfer reaction. Further work will be required to identify which carboxyl group is modified in trans-aconitate.
trans-Aconitate appears to be present in E. coli because we can isolate the radiolabeled methyl ester after incubation of cell extracts with [3H]AdoMet. Its origin, however, is unclear. Wild type E. coli cells are reported to be unable to use trans-aconitate as a carbon source (28), and we are unaware of any pathways where it is a product or substrate for an enzymatic reaction in this organism. We have confirmed that E. coli cells cannot grow on trans-aconitate nor do they contain an aconitate isomerase activity that can convert the citric acid cycle intermediate cis-aconitate to trans-aconitate. Nevertheless, trans-aconitate can be formed spontaneously from cis-aconitate (29-32).
What advantage might the ability to methylate trans-aconitate give E. coli cells? At least in mammalian systems, trans-aconitate is an inhibitor of two central enzymes of the citric acid cycle, aconitase (33, 34) and fumarase (35, 36). The possibility thus exists that the enzymatic methylation of trans-aconitate can attenuate its inhibition of these crucial enzymatic reactions in energy metabolism. This may occur either because the methyl ester of trans-aconitate is inherently less inhibitory to central metabolic reactions or by a novel type of pathway that might convert trans-aconitate methyl ester to a less toxic species. It is even possible that the methylation of trans-aconitate might initiate a pathway that could result in its net conversion to cis-aconitate and its return to the citric acid cycle. These alternatives are presently under investigation in our laboratory. Significantly, the methyltransferase is expressed in early stationary phase when the cessation of rapid cell division may allow altered metabolites to accumulate (see below). Nevertheless, we have been unable to demonstrate a growth phenotype in the methyltransferase-deficient strain we have constructed in this work.
An interesting aspect of trans-aconitate methyltransferase
is that its expression appears to be regulated by the stationary phase
specific factor RpoS (20). Upon starvation, E. coli cells embark upon a developmental program resulting in metabolically less active and more resistant cells (37, 38). The starvation-induced expression of many genes is controlled by RpoS, and an intact rpoS allele is crucial for maintaining cell shape,
resistance to multiple stresses, synthesis of glycogen, and long term
survival in stationary phase cells. The fact that the expression of the trans-aconitate methyltransferase is dependent on the
presence of an intact rpoS gene and the activity of the
enzyme in different growth phases correlates with that of RpoS suggests
that the ability to metabolize trans-aconitate is most
important when cell division is limited and potential inhibitors might
be expected to accumulate. The loss of enzyme activity after extended
stationary phase is likely to reflect both the loss of RpoS protein and
the instability of the enzyme under these conditions, but it is not
clear what the physiological significance of this decrease is.
Although we have detected an active trans-aconitate methyltransferase activity in the yeast S. cerevisiae, no activity has been found in extracts from nematodes or mouse brain. Interestingly, although potential homologous open reading frames have been found in the genomes of a number of procaryotes, there is no clear homolog in the complete genome sequence of yeast. This latter result indicates that this activity may have arisen independently in yeast and bacteria or that the sequence may have diverged rapidly.
In certain plants, trans-aconitate is made in relatively
large amounts and can represent up to 12% of tissue dry weight (39, 40). The enzyme responsible for this conversion is aconitate isomerase
that catalyzes the formation of trans-aconitate from cis-aconitate (27, 41). The function of the accumulation of trans-aconitate in plants is not clear. It does present a
problem for animals that consume it such as ruminants where it is
associated with "grass tetany," a calcium-magnesium deficiency
linked to the chelation properties of a ruminal bacterial metabolite of trans-aconitate, tricarballylate (24, 42).
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ACKNOWLEDGEMENTS |
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We thank Dr. Kym Faull of the UCLA Center for Molecular and Medical Mass Spectroscopy and Dr. Audree Fowler at the UCLA Protein Microsequencing Facility for their expert analyses. We also thank our colleagues Drs. Jon Gary, Jon Lowenson, and Agnieszka Niewmierzycka for their gifts of cell extracts.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM26020.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.
To whom correspondence should be addressed: UCLA Molecular Biology
Institute, Box 951570, Los Angeles, CA 90095-1570. Tel.: 310-825-8754;
Fax: 310-825-1968; E-mail: clarke{at}mbi.ucla.edu.
2 E. coli Genetic Stock Center, Yale University, New Haven, CT. The Web site address is as follows: http://www.cgsc.biology.yale.edu.
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ABBREVIATIONS |
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The abbreviations used are: [14C]AdoMet, S-adenosyl-L-[methyl-14C]methionine; [3H]AdoMet, S-adenosyl-L-[methyl-3H]methionine; AdoMet, S-adenosyl-L-methionine; PCR, polymerase chain reaction; kb, kilobase pair; MES, 2-(N-morpholino)ethanesulfonate; HPLC, high pressure liquid chromatography.
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REFERENCES |
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