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J Biol Chem, Vol. 274, Issue 50, 35407-35414, December 10, 1999
From the The biosynthetic reaction scheme for the
compatible solute mannosylglycerate in Rhodothermus marinus
is proposed based on measurements of the relevant enzymatic activities
in cell-free extracts and in vivo 13C labeling
experiments. The synthesis of mannosylglycerate proceeded via two
alternative pathways; in one of them, GDP mannose was condensed with
D-glycerate to produce mannosylglycerate in a single reaction catalyzed by mannosylglycerate synthase, in the other pathway,
a mannosyl-3-phosphoglycerate synthase catalyzed the conversion of GDP
mannose and D-3-phosphoglycerate into a phosphorylated intermediate, which was subsequently converted to mannosylglycerate by
the action of a phosphatase. The enzyme activities committed to the
synthesis of mannosylglycerate were not influenced by the NaCl
concentration in the growth medium. However, the combined mannosyl-3-phosphoglycerate synthase/phosphatase system required the
addition of NaCl or KCl to the assay mixture for optimal activity. The
mannosylglycerate synthase enzyme was purified and characterized. Based
on partial sequence information, the corresponding mgs gene was identified from a genomic library of R. marinus. In
addition, the mgs gene was overexpressed in
Escherichia coli with a high yield. The enzyme had a
molecular mass of 46,125 Da, and was specific for GDP mannose and
D-glycerate. This is the first report of the characterization of a mannosylglycerate synthase.
Most microorganisms capable of osmotic adaptation accumulate
compatible solutes in response to increases in the levels of salts or
sugars in the environment. Some compatible solutes, such as glutamate,
betaine, and trehalose are widespread in mesophilic organisms, but
compatible solutes unique to thermophiles and
hyperthermophiles, that appear to be associated with thermal
adaptation, have also been identified in recent years. Newly discovered
solutes from thermophilic and hyperthermophilic organisms include
cyclic-2,3-bisphosphoglycerate (1), two isomers of
di-myo-inositolphosphate (2-4), mannosylglycerate and
mannosylglyceramide (3, 5, 6),
di-mannosyl-di-myo-inositolphosphate (4),
diglycerolphosphate (7), and galactosyl-5-hydroxylysine (8). Many of
these solutes have only been identified in marine thermophilic and
hyperthermophilic organisms. The observation that some of these solutes
accumulate at supraoptimal growth temperatures, combined to their
effectiveness in protecting enzymes in vitro supports the
hypothesis that they play a role in thermoprotection of living cells
(2-4, 6-12).
Our knowledge of the biosynthetic pathways for compatible solutes in
prokaryotes has increased significantly in recent years to include the
synthesis of trehalose (13), ectoine (14, 15), glucosylglycerol (16,
17), galactosylglycerol (18), cyclic-2,3-bisphosphoglycerate (19, 20),
and di-myo-inositolphosphate (21, 22). Mannosylglycerate, a
solute initially identified in a few red algae (23), was recently identified in thermophilic bacteria of the genera
Rhodothermus, Thermus (5), Petrotoga
(4), and hyperthermophilic archaea of the genera Pyrococcus
(3), Thermococcus (8), and Methanothermus (7). Mannosylglycerate appears to be one of the most prevalent compatible solutes in thermophilic and hyperthermophilic bacteria and
archaea, and is believed to be, along with
di-myo-inositolphosphate, an archetypal osmolyte of
organisms living near or at the highest growth temperatures for life.
In Rhodothermus marinus, mannosylglycerate accumulates in
response to growth at supraoptimal temperature and salinity while the
amide form, mannosylglyceramide, accumulates exclusively in response to
salt stress (5, 6). Furthermore, mannosylglycerate was found to protect
several enzymes against inactivation by temperature and freeze-drying
(12). The elucidation of metabolic pathways for the synthesis of
compatible solutes is essential to understand the regulatory mechanisms
involved in the adaptation of many thermophilic and hyperthermophilic
organisms to fluctuations in salt or temperature. Moreover, the
biochemical and genetic characterization of key enzymes is also a
prerequisite for achieving overproduction of these compounds in
suitable hosts.
In this study we elucidated the biosynthetic pathways of
mannosylglycerate in R. marinus, and characterized the
enzyme mannosylglycerate synthase, catalyzing a final step in the
synthesis of mannosylglycerate. In addition, we report the cloning and
overexpression of the respective gene in Escherichia
coli.
Strains, Plasmids, and Culture Conditions
The type strain of R. marinus DSM 4252 (Deutsche
Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany)
was grown on Degryse medium containing 2.5 g of tryptone and
2.5 g of yeast extract/liter (24), and supplemented with 1, 4, and 6% NaCl (w/v). Cultures were grown in a 5-liter fermentor at 65 °C,
with continuous gassing with air and stirred at 150 rpm. Cell growth
was monitored by measuring the turbidity at 600 nm.
Escherichia coli TG1 (supE thi hsd Preparation of Cell-free Extracts
Cells were harvested by centrifugation (8,000 × g, 15 min, 20 °C) during the late exponential phase of
growth and frozen at Enzyme Assays
The specific activities of phosphoglucose isomerase (EC
5.3.1.9), phosphomannose isomerase (EC 5.3.1.8), and phosphomannomutase (EC 5.4.2.8) were determined by spectrophotometry at 55 °C using coupled reaction assays. Phosphoglucose isomerase and phosphomannose isomerase assays were based on the method described by Slein (26). Phosphomannomutase activity was assayed by the method described by
Pindar and Bucke (27). Mannose-1-phosphate guanylyltransferase (EC
2.7.7.13), mannosylglycerate synthase, and the combined mannosyl-3-phosphoglycerate synthase/phosphatase activities were assayed by 1H NMR to detect and quantify substrate
consumption and product formation. The assay mixture for measurement of
mannose-1-phosphate guanylyltransferase activity was based on that
suggested by Munch-Peterson (28) and contained 10 mM
MgCl2, 4 mM EDTA, 4 mM
guanosine-5'-triphosphate (Sigma), 4 mM mannose 1-phosphate
(Sigma), and 2 units of inorganic pyrophosphatase in Tris-HCl (20 mM, pH 7.6). For determination of the combined activity of
mannosyl-3-phosphoglycerate synthase/phosphatase the reaction mixture
contained 10 mM MgCl2, 4 mM EDTA, 4 mM GDP mannose (Sigma), 4 mM
D-3-P-glycerate (sodium salt, Sigma), and 0.3 M
KCl in 20 mM Tris-HCl (pH 7.6). Mannosylglycerate synthase was measured in an assay mixture containing 10 mM
MgCl2, 4 mM EDTA, 4 mM GDP mannose,
and 4 mM D-glycerate (hemicalcium salt, Sigma)
in the same buffer.
Discontinuous or continuous methods were used to measure the activities
of these enzymes at 65 °C. In the discontinuous method, cell
extracts (generally, 100 µl) were incubated with the substrates, in a
total reaction volume of 0.6 ml, for various periods of time, and the
reactions were stopped by cooling and acidification with 25 mM HCl. After centrifugation, the pH of the supernatants
was adjusted to 7.0 before freeze-drying. The residues were dissolved in 2H2O and analyzed by NMR. In the continuous
method, the substrates were added to a 5-mm NMR tube and the reactions
started by addition of aliquots of the enzyme preparation. The time
course of product formation was followed by sequential acquisition of
spectra. The reactions were stopped by rapid cooling followed by
acidification. After centrifugation, the supernatant was neutralized
and MG quantified by 1H NMR. Control assays
lacking the cell extracts or the substrates were also carried out. All
specific activities are the mean values of six assays in crude extracts
prepared from cells derived from three independent growths.
NMR Spectroscopy
NMR spectra were acquired on Bruker DRX500 or AMX300
spectrometers. 1H NMR spectra were acquired with a 5-mm
broad band inverse probe head with presaturation of the water signal.
For quantification spectra were acquired with a repetition delay of
35 s and acetate was used as an internal concentration standard.
Proton chemical shifts are relative to
3-(trimethylsilyl)propanesulfonic acid (sodium salt).
Analysis of Mannosylglycerate Synthesis by TLC
Chromatograms were run on silica gel plates (Silica 60; Merck)
with a solvent system composed of chloroform, methanol, 25% ammonia
(6:10:5, v/v). The sugar and sugar derivatives were visualized by
spraying with Purification of the Native Mannosylglycerate Synthase
The enzyme, catalyzing the synthesis of MG from GDP mannose and
D-glycerate, was purified by fast protein liquid
chromatography (Amersham Pharmacia Biotech) at room temperature from
R. marinus cells grown in medium containing 4% NaCl.
Fractions were examined for the presence of the enzyme by the
mannosylglycerate synthase assay method described above and
visualization of MG formation by TLC.
Ion-exchange Chromatography--
Cell-free extract was applied
to a column (XK50/30; bed volume, 250 ml) packed with DEAE-Sepharose
fast flow (Amersham Pharmacia Biotech) equilibrated with Tris-HCl (20 mM, pH 7.6). Elution was carried out with a two-step linear
NaCl gradient (0-0.2 M and 0.2-0.4 M) in the
same buffer. Mannosylglycerate synthase activity was found in the
fractions eluting at approximately 0.3 M NaCl.
Hydrophobic Interaction Chromatography--
Ammonium sulfate was
added to the pooled fractions from the previous step to a final
concentration of 0.5 M. This sample was applied to a column
(XK16/20; bed volume, 90 ml) packed with phenyl-Sepharose (Amersham
Pharmacia Biotech) equilibrated with 50 mM potassium phosphate (pH 7.0), containing 0.5 M ammonium sulfate.
Elution was carried out with a decreasing linear gradient of ammonium sulfate (0.5-0.0 M) in potassium phosphate buffer.
Mannosylglycerate synthase eluted toward the end of the gradient.
Superdex-200 Gel Filtration Chromatography--
The active
fractions were pooled and concentrated by ultrafiltration (30-kDa
cutoff). Fractions (1 ml) were applied to a gel filtration column
(XK16/60; bed volume, 120 ml) packed with Superdex-200 (Amersham
Pharmacia Biotech) equilibrated with 0.15 M NaCl in 50 mM Tris-HCl (pH 7.6), and eluted with the same buffer.
Anion-exchange Chromatography (Resource Q)--
Active fractions
were pooled, concentrated, and equilibrated to 20 mM
Tris-HCl (pH 6.7). The resulting sample was applied to a 6-ml Resource
Q column. Elution was carried out with a two-step linear NaCl gradient
(0-0.2 M and 0.2-1 M). The fractions eluting between 0.22 and 0.25 M NaCl contained mannosylglycerate synthase.
Electroelution of Mannosylglycerate Synthase from a Native
PAGE--
Electrophoresis of native proteins was performed on 7.5%
polyacrylamide (30). Four protein bands were located by cutting two
thin longitudinal strips from the sides of the gel slab and staining
with Coomassie Blue. The four bands were recovered by electrophoretic
elution from the gel using a BIOTRAP BT 1000 (Schleider & Schuell) at
200 V for 4 h, and each protein was assayed for mannosylglycerate
synthase activity. Of these bands, only one had this activity,
originating a single band by SDS-PAGE.
The N-terminal amino acid sequence of mannosylglycerate synthase was
determined by the method of Edman and Begg (31) using an Applied
Biosystem Model 477A protein sequencer. The internal sequences were
determined by Edman degradation after digestion with trypsin and
separation of the peptides by micro-HPLC at the Microchemical Facility,
Emory University School of Medicine, GA.
DNA Methodology, Cloning, and Analysis
Most DNA manipulations followed standard molecular techniques
and procedures (32). Total DNA from R. marinus was purified by the method of Marmur (33). For sequencing purposes, recombinant plasmid DNAs were prepared using plasmid kits (QIAGEN). Southern blots
of restriction endonuclease-cleaved genomic or plasmid DNAs, and colony
blots were hybridized with DNA probes labeled with a digoxigenin DNA
labeling and detection kit (Roche Molecular Biochemicals).
From the N-terminal amino acid sequence of the purified
mannosylglycerate synthase, FPFKHEHPEV (amino acids 6-15), the
degenerate sense primer
5'-TTCCC(G/C)TTCAAGCA-CGAGCACCC(G/C)GAGGT-3' was designed. From
the partial amino acid sequences of three internal peptides, HFYDADIT
(amino acids 97-104), QVELLELFT (amino acids 286-294), and GYDYAQQ
(amino acids 359-366), the degenerate antisense primers
5'-GTGATGTC(G/C)GCGTCGTAGAAGTG-3',
5'-GTGAA(G/C)AGCTC(G/C)AG(G/C)AGCTC(G/C)ACCTG-3', and
5'-TACTGCTG(G/C)GCGTAGTCGTA(G/C)CC-3', respectively, were designed. PCR amplifications were carried out in a
Perkin-Elmer GeneAmp PCR System 2400 in reaction mixtures (50 µl)
containing 200 ng of genomic R. marinus DNA, 100 ng of each
primer, 10 mM Tris-HCl (pH 9.0), 1.5 mM
MgCl2, 50 mM KCl, 0.5 units of Taq
DNA polymerase, and 0.2 mM of each deoxynucleoside
triphosphate (Amersham Pharmacia Biotech). The mixture was preincubated
for 5 min at 95 °C and then subjected to 20 cycles of denaturation
at 95 °C for 1 min. Annealing was performed at gradually decreasing
temperatures (59 to 49 °C) for 1 min, and primer extension was at
72 °C for 1 min, followed by 10 cycles in which the annealing
temperature remained constant at 49 °C. The extension reaction in
the last cycle was prolonged for 5 min. Amplification products were
purified from agarose gels for use as hybridization probes, and ligated to the pGEM-T Easy vector (Promega).
To obtain a genomic library from R. marinus, total DNA was
partially digested with restriction enzyme Sau3A. Fragments
ranging from 1 to 5 kbp were purified from agarose gels, and ligated
into the BamHI site of dephosphorylated pUC18. The ligation
mixture was used to transform E. coli TG1 cells. Transformed
cells were plated on YT-broth supplemented with ampicillin,
5-bromo-4-chloro-3-indolyl- Nucleotide sequences were determined by MWG-BIOTECH (Ebersberg,
Germany) using the LI-COR 4200 automated sequencing system. Inserts of
positive pUC18 clones (pMG721, pMG7, and pMG161), pGEM-T Easy-clones,
and plasmid clone pMG37 were sequenced in both orientations using
vector- and insert-specific oligodeoxynucleotide primers. Nucleic acid
and protein sequence analyses were conducted with programs in the
Wisconsin Genetics Computer Group (GCG) software package (34). The
European Bioinformatics Institute data bases, and functionally
annotated genomes in the Kyoto Encyclopedia of Genes and Genomes, as
well as the archaeal genome sequence data base were screened for
homologies using the (T)FASTA (35, 36) and (T)BLAST (37) algorithms.
Overexpression of the mgs Gene and Purification of the
Enzyme Characterization of the Native and the Recombinant
Mannosylglycerate Synthase
The temperature profile for the activity of mannosylglycerate
synthase was determined between 35 and 95 °C, by using the
discontinuous method described above. The effect of pH on
mannosylglycerate synthase activity was determined at 90 °C in 50 mM MES (pH 5.5-6.5) and 50 mM BisTris-propane
buffer (pH 6.5-9.5). All pH values were measured at room temperature;
pH values at 90 °C were calculated by using
The molecular mass of native mannosylglycerate synthase was determined
on a Superose 12 column (Amersham Pharmacia Biotech) equilibrated with
50 mM sodium phosphate buffer (pH 7.6) containing 0.15 M NaCl. Cytochrome c (12.4 kDa), carbonic
anhydrase (29 kDa), albumin (66 kDa), aldolase (158 kDa), and ferritin
(440 kDa) were used as standards. The isoelectric point of native
mannosylglycerate synthase was determined by isoelectric focusing
(model 111 mini-IEF cell, Bio-Rad), according to the manufacturer. A pH
3-10 isoelectric focusing gel and standards in a range of pI 4.5-9.6
were used.
Nucleotide Sequence Accession Number
The nucleotide sequence of mannosylglycerate synthase has been
deposited in GenBank under accession number AF173987.
Enzymatic Activities Involved in the Synthesis of
Mannosylglycerate--
By analogy with the known biosynthetic pathways
of other sugar derivatives, such as glucosylglycerol (16) and trehalose (38), the terminal reaction in the synthesis of MG should involve a
sugar nucleotide as donor of the glycosyl moiety, and a phosphorylated acceptor (3-P-glycerate). From an array of experiments with GDP mannose, UDP mannose, and ADP mannose as donors, and
D-3-P-glycerate as acceptor, we were unable to detect the
formation of MG in cell extracts prepared from R. marinus
grown in medium containing 4% NaCl. Activity for the synthesis of MG,
however low, was detected upon the addition of NaCl or KCl (150, 300, and 450 mM), to the enzyme assay, when GDP mannose was used
as a glycosyl donor. Optimal activity was observed at 300 mM NaCl or KCl. Unexpectedly, the activity for the
formation of MG in cell extracts was significantly higher when
D-glycerate, instead of D-3-P-glycerate, was
used in addition to GDP mannose (Table I,
compare the values for mannosylglycerate synthase and mannosyl
3-P-glycerate synthase/mannosyl 3-P-glycerate phosphatase).
Furthermore, this activity did not depend on the salt concentration in
the assay mixture (0-450 mM NaCl or KCl). 1H
NMR was used to detect and monitor the time course for the formation of
MG in crude cell extracts at 65 °C. In the anomeric region of the
spectra clear resonances due to the anomeric protons of MG and the
ribose and mannose moieties in GDP mannose were detected, allowing to
monitor on line the consumption of the substrate and build up of the
product (Fig. 1).
The use of two different substrates, D-glycerate and
D-3-P-glycerate, combined with the differences in the salt
dependence, led us to suspect that R. marinus contained two
enzymatic systems for the synthesis of MG. This hypothesis was further
supported by the observation that D-3-P-glycerate was not a
substrate for purified mannosylglycerate synthase, the enzyme
catalyzing the synthesis of mannosylglycerate from GDP mannose and
D-glycerate, and the activity of the enzyme was independent
of the presence of NaCl or KCl. Firm evidence for the salt dependent
activity, which used GDP mannose and D-3-P-glycerate as
substrates, was obtained from experiments performed with the
flow-through of the DEAE-Sepharose column in the chromatographic
procedure for purification of mannosylglycerate synthase. This fraction
did not contain mannosylglycerate synthase activity, as assayed with
GDP mannose and D-glycerate. On the other hand, incubation
of an aliquot with GDP mannose and D-3-P-glycerate, at
65 °C, did not lead to the immediate formation of MG, but to a
compound that remained at the origin of TLC plates, suggesting that a
phosphorylated product had been formed. To confirm this hypothesis,
alkaline phosphatase was added to the reaction mixture after the
initial incubation period with GDP mannose and D-3-P-glycerate. The analysis of the reaction mixture by
TLC revealed the presence of MG (Fig. 2).
The identity of the compound was confirmed by 1H NMR. These
results showed that mannosyl-3-phosphoglycerate synthase activity was
present in the fractions that did not adsorb onto the DEAE-Sepharose
column, and demonstrated the presence of an enzymatic system for MG
synthesis, that did not involve mannosylglycerate synthase. Evidence
for the presence of a phosphatase, which acted upon
mannosyl-3-phosphoglycerate in cell extracts, was further obtained from
the decrease in the formation of MG when cell extracts were treated
with the phosphatase inhibitor, sodium fluoride (results not
shown).
To study the effect of salinity of the growth medium on the expression
of the enzymes involved in the biosynthetic pathway of MG, cell
extracts from R. marinus grown in medium containing 1, 4, and 6% NaCl were examined, and the enzymatic activities responsible
for catalysis of the sequence of reactions from glucose 6-phosphate to
the formation of GDP mannose, as well as those catalyzing the final
step in the biosynthesis of MG, were determined. The enzymes committed
to the synthesis of MG were not affected by the salinity of the growth
medium, but the three activities involved in the formation of
mannose-1-P from glucose-6-P decreased significantly. However, all the
activities were constitutive under the conditions tested (Table
I).
Synthesis of Mannosylglycerate in Whole Cells from
13C-Labeled Glucose--
The constitutive character of the
enzymes involved in the synthesis of MG was also apparent from the
immediate synthesis of this solute observed in non-growing cell
suspensions of R. marinus in response to a salt upshock. The
production of MG by cells of R. marinus was investigated by
in vivo 13C NMR, in experiments where
[1-13C]glucose was supplied to cells grown at 65 °C in
a defined medium containing 1% NaCl and glucose as the only carbon
source. Cells were suspended in phosphate buffer (20 mM, pH
7.5) containing 1% NaCl. Under these conditions labeling of MG could
not be detected; however, increasing the NaCl concentration to 3%
resulted in the appearance of resonances due to C1 (at 98.7 ppm) and C3 (at 63.3 ppm) of the mannose and glycerate
moieties of MG, respectively (spectra not shown). The MG content
increased in the first 50 min after the salt upshock and remained
constant thereafter. These data also show that the mannose moiety is
derived directly from glucose, and glycolysis proceeds via the
Embden-Meyerhoff pathway in R. marinus since label from
[1-13C]glucose ends on carbon 3 of glycerate (39).
Purification and Sequence Analysis of Mannosylglycerate
Synthase--
The enzyme, catalyzing the synthesis of
mannosylglycerate from GDP mannose and D-glycerate was
purified using four chromatographic steps described under "Materials
and Methods." At this stage, approximately 30% of the
mannosylglycerate synthase activity present in the initial crude
extract was recovered. This preparation contained four bands in native
PAGE (Fig. 3). The enzyme was obtained by electroelution and migrated as a single band in SDS-PAGE with an
apparent molecular mass of 45 kDa. A value of 122 ± 9 kDa was found by gel filtration at room temperature. The isoelectric point was
4.8. The N-terminal amino acid sequence of the purified enzyme and
those of five peptides generated by a tryptic digestion were determined
to be SLVVFPFKHEHPEVLLHNVRVAAXHPRXH, IHFYDADITSFGPD, ASTDAMITWMITR,
LYGGLDD, QVELLELFTTPVR, and GYDYAQQYLYR, respectively.
Cloning, Sequencing and Overexpression of mgs Gene in E. coli--
PCR amplification from the genomic DNA of R. marinus with primers based on the N-terminal and three internal
amino acid sequences yielded unique products with lengths of
approximately 300, 900, and 1100 nucleotides. The nucleotide sequence
of the 1100-bp fragment predicted an open reading frame encoding a
peptide of 360 amino acids, which contained the N-terminal sequence as
well as all internal, partial sequences of mannosylglycerate synthase.
This fragment was used as a probe to isolate clones from the
constructed genomic libraries of R. marinus. In total, three
positive clones were isolated. One plasmid derived from the genomic
library, designated pMG721, contained a 3.4-kbp partial
Sau3A fragment, whereas the two plasmids of the size
selected library, designated pMG7 and pMG161, contained each
orientation of the expected 2.1-kbp PstI fragment.
Nucleotide sequence analysis showed, that the 3.4-kbp fragment
overlapped the 2.1-kbp fragment almost completely (Fig. 4).
Several open reading frames were found on both strands (Fig. 4). An
open reading frame, designated mgs, encoding a protein of
397 amino acids, was found showing a perfect correlation with the
sequence of the 1100-bp PCR fragment used as probe. The ATG codon (at
position 689) functions as initiation codon for the mgs gene
(Fig. 5), since it was directly followed
by a stretch of 23 amino acids identical to the N-terminal sequence of
the purified enzyme. A putative ribosome binding sequence is positioned 7-12 bp upstream of the ATG codon. The molecular mass based on the
deduced amino sequence was calculated to be 46,125 Da, which is in
excellent agreement with the value of 45 kDa obtained by SDS-PAGE with
the native enzyme. Comparison of the amino acid sequence of
mannosylglycerate synthase with sequences in several data bases showed
only low similarities. The highest similarity (29% identity) was found
with a 382-amino acid long hypothetical protein of Pyrococcus
horikoshii.
Two open reading frames located directly upstream and downstream of the
mgs gene with lengths of 219 and 308 amino acids, respectively, did not show any significant similarities with other known sequences in the data bases. However, in the opposite orientation upstream of the mgs gene, an open reading frame (with an ATG
start codon at position 382) encodes a 120-amino acid partial sequence with high similarity (47% identity) to a glucosamine-6-phosphate deaminase of E. coli (40). The 3' end of the cloned region
(position 2687 to end) was identical to a previously cloned sequence of the lig gene coding for the DNA ligase of R. marinus (41).
For overexpression in E. coli, the PCR-amplified
mgs coding sequence was cloned under the control of the
strong inducible tac promoter in pKK223-3. The sequence of
the plasmid insert was identical to that of the native gene. Moreover,
the N-terminal sequence of the recombinant protein was also identical
to the native N-terminal sequence (data not shown). SDS-PAGE analysis of crude extracts from E. coli XL1-Blue (pMG37) grown in the
presence of IPTG revealed the presence of an extra band of 45 kDa, that was not observed in crude extracts from E. coli XL1-Blue
(pKK223-3) (Fig. 6). A heat treatment at
75 °C for 30 min resulted in an extensive purification of the crude
extract with the 45-kDa band being almost exclusively present (Fig. 6).
Incubation of the crude extract from E. coli XL1-Blue
(pMG37) at 80 °C with GDP mannose and D-glycerate
resulted in the formation of MG, but this compound was not detected
after incubation of those substrates with a crude extract from E. coli XL1-Blue (pKK223-3). The specific activity of
mannosylglycerate synthase in crude extracts of E. coli
XL1-Blue (pMG37) was 10 µmol/min/mg of protein at 90 °C.
Catalytic Properties of Mannosylglycerate Synthase--
The native
enzyme preparation obtained by electroelution had a specific activity
of 70 µmol/min/mg of protein. The enzyme did not have detectable
activity for the backward reaction. The substrate specificity of
mannosylglycerate synthase was investigated using GDP mannose, ADP
mannose, UDP mannose, mannose 1-phosphate, and mannose 6-phosphate as
sugar donors, and D-glycerate, L-glycerate, and
D-3-P-glycerate as sugar acceptors. To investigate the
ability of mannosylglycerate synthase to catalyze the synthesis of
other glycosylconjugates, GDP glucose, UDP glucose, and ADP glucose were tested as sugar donors, and glycerol 3-phosphate, glucose 6-phosphate, glucose 1-phosphate, and glucose as sugar acceptors. The
enzyme was specific for GDP mannose and D-glycerate. An
absolute stereospecificity for D-glycerate was found. UDP
mannose was the only sugar nucleotide, other than GDP mannose, that was
used by the enzyme, but to a very low extent (less than 3% of that of GDP mannose). Km values were determined for GDP
mannose and D-glycerate (Table
II). NaCl and KCl, in the concentration range of 50-500 mM, had no effect on the enzyme activity.
On the other hand, addition of Mg2+ was required for
maximal activity; the activity of mannosylglycerate synthase was
2.5-fold lower in the absence of this divalent cation. The activity was
undetectable at room temperature, and maximal activity was reached
between 85 and 90 °C (Fig. 7), which
is well above the temperature range for growth of R. marinus
(6). From the linear part of an Arrhenius plot, the activation energy
of 63 kJ/mol was calculated. At the temperature required for maximal activity, 90 °C, the stability of the enzyme was only moderate, 50%
of the activity being lost after 30 min incubation (Fig.
8). At 65 °C the specific enzymatic
activity was 5-fold lower than the maximum value, and the half-life for
inactivation increased to 170 min. The enzyme thermostability was not
improved by Ca2+ (5 mM), nor was it enhanced
under anaerobic conditions. Within the pH range examined (5.5-8.5),
the activity of the enzyme at 90 °C remained nearly constant between
pH 5.5 and 7.0, but decreased as the pH was increased above 7.0 (Fig.
9). These properties were determined
using a partially purified enzyme preparation with a specific activity
of 13 µmol/min/mg of protein.
The temperature profile for activity (Fig. 7), the thermostability at
65 and 90 °C (Fig. 8) as well as the kinetic parameters Km and Vmax were also
determined for the recombinant enzyme (Table II). Overall, the
properties of the native and recombinant enzyme were similar, but the
recombinant form was less thermostable.
In this study, two alternative routes for the synthesis of the
compatible solute mannosylglycerate in R. marinus are
proposed based on measurements of the relevant enzymatic activities in cell-free extracts and in vivo 13C labeling
experiments (Fig. 10). In R. marinus GDP mannose is used as the glycosyl donor for the
synthesis of MG, in accordance with the general observation that GTP is
the preferred substrate to carry mannose in an activated form in many
reactions (42). From GDP mannose, the synthesis proceeds via a branched
pathway: a single-step reaction converts GDP mannose and
D-glycerate to MG by the action of mannosylglycerate
synthase; in the other pathway, GDP mannose and
D-3-P-glycerate react to produce the phosphorylated intermediate, mannosyl 3-phosphoglycerate, which is subsequently dephosphorylated by a phosphatase yielding MG.
A noticeable feature distinguishing the two enzymatic systems involved
in the synthesis of MG is their dependence on salt; while the
mannosylglycerate synthase reaction is salt-independent, addition of
NaCl or KCl is required to achieve full activity of the
mannosyl-3-phosphoglycerate synthase/phosphatase system. This salt
dependence is actually a feature common to the biosynthetic pathways of
other osmolytes such as, trehalose, glucosylglycerol, and
galactosylglycerol, whose synthesis also occurs by two-step reactions,
involving the concerted action of synthases and phosphatases (16, 18,
38). For instance, the synthesis of glucosylglycerol involves the
condensation of ADP glucose and glycerol 3-phosphate to produce
glucosylglycerol phosphate, which is subsequently dephosphorylated to
yield glucosylglycerol (16). The enzymes involved in these biosynthetic
pathways have not been purified or thoroughly characterized, but it has
been shown that enzyme activities in crude extracts are strongly
activated by potassium and sodium salts (16-18, 38).
It is, therefore, intriguing that the salt-dependent
enzymatic system for the synthesis of MG has an activity considerably lower (6-14-fold) than that of the salt-independent system, especially if we take into consideration that the concentration of
mannosylglycerate in R. marinus cells is markedly dependent
on the salinity of the growth medium (5, 6). The present study does not
allow an evaluation of the relative contributions of the two pathways
in vivo; however, the in vivo 13C NMR
experiments showed synthesis of MG in R. marinus in response to salt upshock, an observation that seems to support the relevant contribution of the salt-dependent system, at least under
the conditions examined. Also curious is the observation that the enzymatic activities involved in the synthesis of MG, as determined in
cell extracts, were independent of the salt concentration of the growth
medium, whereas salt-induced de novo synthesis of enzymes involved in the formation of several osmolytes has been reported (13,
15, 17, 38). However, our results do not rule out the existence of a
salt-dependent control mechanism at the level of gene
expression (transcription or translation).
The reasons underlying the existence of two distinct pathways for the
synthesis of MG in R. marinus remain unclear, but it is
conceivable that these systems are differentially regulated by salt and
temperature, the two physicochemical parameters that are known to
influence the intracellular concentrations of MG in R. marinus (6). The two pathways could also have different metabolic
functions, one being implicated in the synthesis of MG and the other in
its catabolism, as observed in the synthesis of sucrose in plants,
which employs two different pathways, both involving UDP glucose as
glucosyl donor, but differing in the nature of the glycosyl acceptor:
fructose-6-P or D-fructose. In this case it has been
suggested that sucrose phosphate synthase is primarily involved in the
synthesis of sucrose, whereas sucrose synthase is implicated in sucrose
catabolism (43, 44). However, mannosylglycerate synthase does not
appear to be involved in catabolism of MG since the activity of the
enzyme in the catalysis of the cleavage of MG was not detectable.
In R. marinus GDP mannose is formed via a conventional
pathway involved in the synthesis of polymers containing mannose or mannose derivatives, as observed in the biosynthetic pathway of exopolysaccharides (45, 46) and in colanic acid synthesis (47). The key
glycolytic metabolite, glucose 6-phosphate is converted to mannose
6-phosphate by the action of phosphohexose isomerases, and mannose
6-phosphate is subsequently converted to mannose 1-phosphate by
phosphomannomutase. The activated sugar, mannose 1-phosphate, and GTP
are the substrates for mannose-1-phosphate guanylyltransferase
catalyzing the formation of GDP mannose and pyrophosphate (Fig. 10). A
marked difference exists in the magnitude of the specific activities of
the enzymes involved in the formation of GDP mannose in R. marinus and those committed to the synthesis of MG, as generally
observed when comparing primary metabolic trunks with specific branches.
In this study we choose to characterize biochemically and genetically
the enzyme system that showed highest in vitro activity for
the synthesis of mannosylglycerate, mannosylglycerate synthase. The
enzyme has a high degree of specificity with regard to the substrates
GDP mannose and D-glycerate. It is interesting to note that
lactose synthase, one of the most extensively characterized glycosyltransferases, also uses a non-phosphorylated glycosyl acceptor.
The enzyme catalyzes the transfer of galactose from UDP galactose to
D-glucose, in the presence of The properties of the native and recombinant mannosylglycerate synthase
are very similar, except for a lower thermal stability of the
recombinant enzyme at 90 °C, which may be accounted by slight
differences in the folding of the protein produced in the heterologous
host. Mannosylglycerate synthase shares a low degree of amino acid
sequence homology with other sugar transferases and functionally
related enzymes. Moreover, comparison of the amino acid sequence of
mannosylglycerate with those in several data bases showed only very low
similarities, although mannosylglycerate is synthesized by several
hyperthermophilic archaea, such as Pyrococcus furiosus and
P. horikoshii, whose complete genomic sequences are known.
In conclusion, we believe that the results reported in this study on
the characterization of the biosynthesis of MG in the thermophilic
bacterium R. marinus, as well as of the key enzyme, mannosylglycerate synthase, represent an important step toward the
elucidation of MG synthesis and regulation in hyperthermophilic archaea
sharing with R. marinus the ability to accumulate this osmolyte.
*
This work was supported in part by the European Community
Biotech Program Extremophiles as Cell Factories, BIO4-CT96-0488, and by
PRAXIS XXI and FEDER, Portugal PRAXIS/2/2.1/BIO/1109/95.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 GenBankTM/EMBL Data Bank with accession number(s) AF173987.
§
Supported by Postdoctoral Fellowship BPD/6049/95 from PRAXIS XXI.
**
To whom correspondence should be addressed: Instituto de Tecnologia
Química e Biológica, Apartado 127, 2780 Oeiras, Portugal. Tel.: 351-1-4469800; Fax: 351-1-4428766; E-mail:
santos@itqb.unl.pt.
The abbreviations used are:
IPTG, isopropyl-
Biosynthesis of Mannosylglycerate in the Thermophilic
Bacterium Rhodothermus marinus
BIOCHEMICAL AND GENETIC CHARACTERIZATION OF A MANNOSYLGLYCERATE
SYNTHASE*
§,¶,
,**
Instituto de Tecnologia Química e
Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, Apartado 127, 2780 Oeiras, Portugal and the ¶ Departamento de
Bioquímica and Centro de Neurociências de Coimbra,
Universidade de Coimbra, 3000 Coimbra, Portugal
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5
(lac-proAB) [F'traD36 proAB
lacIqZM15]) and E. coli
XL1-Blue (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac
[F'proAB lacIqZM15
Tn10 (Tetr)]) were used as hosts for the
cloning vectors pUC18, pGEM-T Easy (Promega), and pKK223-3 (Amersham
Pharmacia Biotech). E. coli was grown in YT-medium
containing 10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl/liter. Ampicillin was added at a final concentration of 100 µg/ml for selection of plasmids.
5-Bromo-4-chloro-3-indolyl-
-D-galactopyranoside and
IPTG1 were obtained from
Roche Molecular Biochemicals (Germany) and added at a final
concentration of 80 µg/ml and 0.5 mM, respectively.
80 °C. The cell sediment was suspended in
Tris-HCl (20 mM, pH 7.6) containing DNase I (10 µg/ml
extract) and MgCl2 (5 mM), and a mixture of
protease inhibitors: phenylmethylsulfonyl fluoride (0.08 mg/ml), leupeptine (0.02 mg/ml), and antipain (0.02 mg/ml). Cells were
disrupted in a French pressure cell, followed by centrifugation (20,000 × g, 1 h, 10 °C) to remove cell
debris. Cell extracts were applied to a Sephadex G-25 column (Amersham
Pharmacia Biotech), equilibrated with 20 mM Tris-HCl (pH
7.6) to remove mannosylglycerate (MG) and other low molecular weight
compounds prior to measuring enzyme activities. The protein content was
determined by the Bradford assay (25) using bovine serum albumin as standard.
-naphtol-sulfuric acid solution followed by charring
at 120 °C (29). Authentic standards of mannose, mannose 1-phosphate,
GDP mannose, GDP, GMP, guanosine, and mannosylglycerate were used for
comparative purposes.
-D-galactopyranoside, and
IPTG. Approximately, 2 × 104 transformants were
obtained after 18 h of growth at 37 °C. Similarly, a partial
genomic library from R. marinus, containing DNA fragments selected by Southern analysis of genomic DNA preparations with the
mgs probe, was obtained by complete digestion of total DNA with restriction enzyme PstI, followed by ligation of
purified fragments of about 2.1 kbp in size into the PstI
site of pUC18, and subsequent transformation of E. coli
XL1-Blue cells with the ligation mixture. Positive clones were detected
by colony hybridization with digoxigenin-labeled probes as described above.
The mgs gene was amplified by PCR using
the forward primer 5'-GCGGAATTCATGAGCCTGGTCGTTTTTCCC-3' with an additional EcoRI recognition sequence (underlined)
immediately upstream of the ATG start codon, and the reversed primer
5'-GCGCTGCAGGGATCCTCAGGCGGTCGACAGTGCC-3' with additional
PstI and BamHI recognition sequences (underlined) directly behind the TGA stop codon. The PCR product was purified after
digestion with EcoRI and PstI, and ligated into
the corresponding sites in the pKK223-3 expression vector to obtain
plasmid pMG37. E. coli XL1-Blue cells containing pMG37 were
grown to the midexponential growth phase (OD600 = 0.4),
induced with IPTG, and growth continued for a further 3 to 4 h.
Cells were harvested and treated as described above for the preparation
of cell-free extracts. The resulting extract was incubated for 30 min
at 75 °C and centrifuged. After dialysis against 20 mM
Tris-HCl (pH 7.6), the sample was loaded onto a Mono Q (Amersham
Pharmacia Biotech) column that was eluted by a linear gradient of NaCl
(0-500 mM). Fractions with activity were pooled,
concentrated, and applied to a Superdex-200 (Amersham Pharmacia
Biotech) column.
pKa/T °C = 0.011 and 0.015 for MES
and BisTris-propane, respectively. Enzyme thermostability was
determined at 65 and 90 °C by incubating an enzyme solution in 20 mM Tris-HCl (pH 7.6). At appropriate times, samples were
withdrawn and immediately examined for residual mannosylglycerate
synthase activity using the discontinuous assay at 90 °C. Kinetic
parameters were determined at 90 °C. Reaction mixtures contained GDP
mannose (0.165-10.0 mM) plus 10 mM
D-glycerate, or D-glycerate (0.188-10.0
mM) plus 10 mM GDP mannose. Samples were
preheated for 3 min, the reaction was initiated by the addition of the
enzyme preparation, and MG was quantified by 1H NMR.
Experiments were performed in duplicate. Values for
Vmax and Km were determined
from Hanes plots.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Enzymatic activities involved in the biosynthesis of mannosylglycerate
in cell-free extracts of Rhodothermus marinus
View larger version (21K):
[in a new window]
Fig. 1.
Time course for the formation of
mannosylglycerate from GDP mannose and D-glycerate by a
cell extract of R. marinus at 65 °C as monitored by
1H NMR. Assignments of resonances: anomeric protons in
the ribosyl ( 
) and mannosyl moieties (
) in GDP mannose, and
anomeric proton of mannosylglycerate (
).
View larger version (18K):
[in a new window]
Fig. 2.
TLC analysis of reaction products obtained
from different enzymatic reactions. A, reaction
products of purified mannosylglycerate synthase using GDP mannose and
D-glycerate as substrates before (lane 2) and
after (lane 3) treatment with alkaline phosphatase (1 h at
37 °C). B, reaction products from partially purified
mannosyl-3-phosphoglycerate synthase using GDP mannose and
D-3-P-glycerate as substrates before (lane 4)
and after (lane 5) treatment with alkaline phosphatase (1 h
at 37 °C). Mannosylglycerate and guanosine standards are shown in
lanes 1 and 6, respectively.
View larger version (50K):
[in a new window]
Fig. 3.
Native PAGE and SDS-PAGE of mannosylglycerate
synthase. A, four protein bands were found by native
PAGE (7.5%, w/v, acrylamide) after the chromatographic purification
steps (total protein applied was 20 µg). The band indicated by the
arrow had mannosylglycerate synthase activity and was
recovered by electroelution. B, SDS-PAGE of the
electroeluted band (protein applied was 10 µg) gave rise to a single
band (lane 2). Lane 1, molecular mass
markers.
View larger version (10K):
[in a new window]
Fig. 4.
Physical and genetic map of the
mannosylglycerate synthase gene (mgs) of R. marinus. Open reading frames (orf) are
indicated by arrows. The horizontal lines
represent the inserts of the isolated plasmids. NagB'
indicates a partial open reading frame with homology to the
glucosamine-6-phosphate deaminase (NagB) from E. coli (40). Lig' represents the (partial)
lig gene from R. marinus (41).
View larger version (61K):
[in a new window]
Fig. 5.
Nucleotide sequence and deduced amino acid
sequence of the mannosylglycerate synthase gene. The
underlined amino acid sequences indicate the N terminus of
the purified mannosylglycerate synthase and the peptides whose
sequences were determined after tryptic digestion of the purified
mannosylglycerate synthase. The putative ribosome binding sites are
highlighted with a black background.
View larger version (104K):
[in a new window]
Fig. 6.
SDS-PAGE analysis of mannosylglycerate
synthase overproduction in E. coli. Lane
1, molecular mass markers. Lane 2, 5 µl of crude
extract of an IPTG-induced XL1-Blue (pKK223-3) culture. Lane
3, 5 µl of crude extract of an IPTG-induced XL1-Blue (pMG37)
culture. Lane 4, 15 µl supernatant of a heat-treated (30 min at 75 °C) crude extract of an IPTG-induced XL1-Blue (pKK223-3)
culture. Lane 5, 15 µl supernatant of a heat-treated (30 min at 75 °C) crude extract of an IPTG-induced XL1-Blue (pMG37)
culture.
Kinetic parameters for the two substrates of R. marinus
mannosylglycerate synthase and effect of Mg2+ ions
View larger version (13K):
[in a new window]
Fig. 7.
Temperature dependence of native ( ) and
recombinant (
) mannosylglycerate synthase. The enzyme activity
was determined between 35 and 95 °C under the assay conditions
described under "Materials and Methods."
View larger version (12K):
[in a new window]
Fig. 8.
Thermostability of native ( , ) and
recombinant (
, 
) mannosylglycerate synthase.
Mannosylglycerate synthase was incubated at 65 °C (solid
symbols) or at 90 °C (open symbols). Samples were
withdrawn and tested for activity at 90 °C. The half-life for
thermal inactivation at 90 °C of the native and recombinant enzymes
was 30 and 17 min, respectively, at 65 °C both forms of the enzyme
showed identical thermal stability (170 min).
View larger version (7K):
[in a new window]
Fig. 9.
pH dependence of native mannosylglycerate
synthase. The activity of mannosylglycerate synthase was
determined in 50 mM MES ( ) or 50 mM
BisTris-propane () at 90 °C.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 10.
Proposed pathway for the synthesis of
mannosylglycerate in R. marinus. 1, phosphoglucose isomerase; 2, phosphomannose isomerase;
3, phosphomannose mutase; 4, mannose-1-phosphate
guanylyltransferase; 5, mannosylglycerate synthase;
6, mannosyl-3-phosphoglycerate synthase; 7, mannosyl-3-phosphoglycerate phosphatase.
-lactalbumin, with a high
degree of substrate specificity (48, 49). However, this is not a
general feature among glycosyltransferases.
FOOTNOTES
Supported by invited Scientist Fellowship BCC/11978 from
PRAXIS XXI.
ABBREVIATIONS
-D-thiogalactopyranoside;
D-3-P-glycerate, D-3-phosphoglycerate;
MG, mannosylglycerate;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain reaction;
MES, 2-(N-morpholino)ethanesulfonic acid;
kbp, kilobase pair(s);
BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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 C. Neves, M. S. da Costa, and H. Santos Compatible Solutes of the Hyperthermophile Palaeococcus ferrophilus: Osmoadaptation and Thermoadaptation in the Order Thermococcales Appl. Envir. Microbiol., December 1, 2005; 71(12): 8091 - 8098. [Abstract] [Full Text] [PDF]
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S. Alarico, N. Empadinhas, C. Simoes, Z. Silva, A. Henne, A. Mingote, H. Santos, and M. S. da Costa Distribution of Genes for Synthesis of Trehalose and Mannosylglycerate in Thermus spp. and Direct Correlation of These Genes with Halotolerance Appl. Envir. Microbiol., May 1, 2005; 71(5): 2460 - 2466. [Abstract] [Full Text] [PDF]
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N. Empadinhas, L. Albuquerque, J. Costa, S. H. Zinder, M. A. S. Santos, H. Santos, and M. S. da Costa A Gene from the Mesophilic Bacterium Dehalococcoides ethenogenes Encodes a Novel Mannosylglycerate Synthase J. Bacteriol., July 1, 2004; 186(13): 4075 - 4084. [Abstract] [Full Text] [PDF]
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 N. Borges, J. D. Marugg, N. Empadinhas, M. S. d. Costa, and H. Santos Specialized Roles of the Two Pathways for the Synthesis of Mannosylglycerate in Osmoadaptation and Thermoadaptation of Rhodothermus marinus J. Biol. Chem., March 12, 2004; 279(11): 9892 - 9898. [Abstract] [Full Text] [PDF]
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N. Empadinhas, L. Albuquerque, A. Henne, H. Santos, and M. S. d. Costa The Bacterium Thermus thermophilus, Like Hyperthermophilic Archaea, Uses a Two-Step Pathway for the Synthesis of Mannosylglycerate Appl. Envir. Microbiol., June 1, 2003; 69(6): 3272 - 3279. [Abstract] [Full Text] [PDF]
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J. Prasad, P. McJarrow, and P. Gopal Heat and Osmotic Stress Responses of Probiotic Lactobacillus rhamnosus HN001 (DR20) in Relation to Viability after Drying Appl. Envir. Microbiol., February 1, 2003; 69(2): 917 - 925. [Abstract] [Full Text] [PDF]
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M.-M. Sampaio, H. Santos, and W. Boos Synthesis of GDP-Mannose and Mannosylglycerate from Labeled Mannose by Genetically Engineered Escherichia coli without Loss of Specific Isotopic Enrichment Appl. Envir. Microbiol., January 1, 2003; 69(1): 233 - 240. [Abstract] [Full Text] [PDF]
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N. Empadinhas, J. D. Marugg, N. Borges, H. Santos, and M. S. da Costa Pathway for the Synthesis of Mannosylglycerate in the Hyperthermophilic Archaeon Pyrococcus horikoshii. BIOCHEMICAL AND GENETIC CHARACTERIZATION OF KEY ENZYMES J. Biol. Chem., November 16, 2001; 276(47): 43580 - 43588. [Abstract] [Full Text] [PDF]
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