From the Zentrum für Ultrastrukturforschung und
Ludwig Boltzmann-Institut für Molekulare Nanotechnologie,
Universität für Bodenkultur Wien, Gregor-Mendel-Strasse 33, A-1180 Wien, Austria and the § Institut für Chemie,
Universität für Bodenkultur Wien, Muthgasse 18, A-1190 Wien, Austria
Received for publication, January 16, 2001, and in revised form, March 27, 2001
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ABSTRACT |
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The glycan chain repeats of the
S-layer glycoprotein of Aneurinibacillus thermoaerophilus
DSM 10155 contain
D-glycero-D-manno-heptose, which has also been described as constituent of lipopolysaccharide cores of Gram-negative bacteria. The four genes required for
biosynthesis of the nucleotide-activated form
GDP-D-glycero-D-manno-heptose were cloned, sequenced, and overexpressed in Escherichia
coli, and the corresponding enzymes GmhA, GmhB, GmhC, and GmhD
were purified to homogeneity. The isomerase GmhA catalyzed the
conversion of D-sedoheptulose 7-phosphate to
D-glycero-D-manno-heptose
7-phosphate, and the phosphokinase GmhB added a phosphate group to form
D-glycero-D-manno-heptose 1,7-bisphosphate. The phosphatase GmhC removed the phosphate in the C-7
position, and the intermediate
D-glycero- The cell surface of many archaea and bacteria is composed of
crystalline two-dimensional protein arrays, termed
S-layers1 (1). Frequently,
the S-layer proteins are glycosylated (2). The S-layer glycoprotein of
the Gram-positive bacterium Aneurinibacillus thermoaerophilus DSM 10155, a member of the Bacillus/Clostridium group, is composed of disaccharide repeating units of
L,D-Heptose is a common constituent of the LPS
inner core of enteric and non-enteric bacteria (6).
D,D-Heptose, on the other hand, has been
described as component of the outer core region of LPS. Numerous
bacteria, most importantly the pathogens Proteus vulgaris
R110/1959 (7), Haemophilus ducreyi (8), Klebsiella
pneumoniae ssp. pneumoniae R20
(O1 The inner core oligosaccharide of most LPS-containing bacteria consists
of 3-deoxy-D-manno-oct-2-ulosonic acid and
L,D-heptose units. The outer core, however, is
composed predominantly of hexoses and hexosamines (13), but
D,D-heptose has also been reported recently
(see above). Impairment of
3-deoxy-D-manno-oct-2-ulosonic acid biosynthesis
usually results in nonviable cells (6). On the other hand, mutants
having a defect in the biosynthesis of L,D-heptose are viable, albeit with certain
characteristics, referred to as the "deep rough" phenotype in
Escherichia coli and Salmonella typhimurium.
Transduction by the P1 bacteriophage and F-plasmid conjugation are
impaired (14, 15), and the mutants show increased sensitivity to
detergents, bile salts and hydrophobic antibiotics (16). Reduction of
virulence has been reported for Haemophilus influenzae (17)
and S. typhimurium (18). Crystallization of the biosynthesis
enzymes could lead to the development of enzyme inhibitors via
molecular modeling. These inhibitors can support novel antibiotic
therapies to circumvent drug resistance.
The biosynthesis of the nucleotide-activated precursor of
3-deoxy-D-manno-oct-2-ulosonic acid, namely
CMP-3-deoxy-D-manno-oct-2-ulosonic acid, has
been described previously (13). In contrast, the complete biosynthesis
of the nucleotide-activated form of
D/L,D-heptose has not yet been
elucidated. Eidels and Osborn (19) have proposed a four-step pathway
for the synthesis of NDP-L,D-heptose. (i) D-Sedoheptulose 7-phosphate is converted to
D,D-heptose 7-phosphate by phosphoheptose
isomerase; (ii) a mutase catalyzes the second step to form
D,D-heptose 1-phosphate; (iii) this
intermediate product is activated to
NDP-D,D-heptose by the action of NDP-heptose synthetase; (iv) in the last step, an epimerase catalyzes the formation
of the final product NDP-L,D-heptose. The
nucleotide-activated sugar GDP-D,D-heptose has
been described in bakers' yeast (20), and both
ADP-D,D-heptose and
ADP-L,D-heptose have been isolated from
Shigella sonnei and Salmonella minnesota mutants
(21, 22). Recently, it has been shown that heptosyltransferases I and
II from E. coli accept
ADP-L- The first step in heptose biosynthesis was described in S. typhimurium (24), and the phosphoheptose isomerase gene
gmhA from E. coli was cloned and sequenced (25).
So far, no gene product that catalyzes the proposed mutase step has
been described. The RfaE protein, also referred to as ADP-heptose
synthetase, is proposed to be involved in the nucleotide-activating
step. This enzyme has been shown to consist of two separate domains in
E. coli (26). Domain I displays homology to members of the ribokinase family, whereas domain II is homologous to various cytidyltransferases. Thus, RfaE is supposed to be a bifunctional enzyme
involved in the biosynthesis of D,D-heptose
1-phosphate as well as in the nucleotide-activating reaction step. The
rfaD gene product has been proposed to carry out the last
step in the biosynthesis of the L,D-form,
namely the epimerization to yield NDP-L,D-heptose (27). Recently, RfaD has been
crystallized and characterized (28). So far, functional studies have
only been performed for the isomerization reaction (24, 25) and for the
epimerization step (29). Enzymes that might carry out the mutase
reaction, or an alternative phosphatase, have not been described.
In this report we show for the first time the overall biosynthesis of a
nucleotide-activated form of D,D-heptose,
namely GDP-D- Materials--
ATP, GTP, D-sedoheptulose
7-phosphate, and dithiothreitol were obtained from Sigma (Sigma Aldrich
Fluka GmbH, Wien, Austria). D-glycero- Bacterial Strains, Cloning Vectors, and Growth
Conditions--
The E. coli and A. thermoaerophilus strains and cloning vectors used in this study
are listed in Table I. A. thermoaerophilus DSM 10155 was
grown in SVIII medium at 55 °C. E. coli DH5 Analytical Techniques--
Nucleotide-activated sugars were
analyzed on a CarboPac PA-1 column (Dionex, Sunnyvale, CA) using the
method of Palmieri et al. (30) with slight modifications.
Monosaccharides were analyzed on a CarboPac PA-1 column as described
previously (4). Monosaccharide phosphates were analyzed on the
CarboPac PA-1 column using a NaOAc-gradient (100-500 mM)
in 100 mM NaOH. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis was performed according to the original method of
Laemmli (31) using slight modifications. Gels were stained with
Coomassie Brilliant Blue R250. Protein concentrations were determined
by the method of Bradford (32).
Sequence Analysis--
BLAST (33) and MultAlin (34) were used to
analyze nucleotide and protein sequences.
DNA Manipulations, PCR, and DNA Sequencing--
All standard DNA
recombinant procedures were performed according to the methods
described by Sambrook et al. (35) or as recommended by the
corresponding manufacturer. PCR was carried out using a PCR Sprint
thermocycler (Hybaid, Ashford, United Kingdom). DNA sequencing was
performed by MWG BIOTECH (Ebersberg, Germany) and AGOWA (Berlin, Germany).
Cloning of the Heptose Genes--
In order to clone
the genes involved in S-layer glycoprotein glycan biosynthesis,
particularly the dTDP-L-rhamnose genes, an alignment of 14 RmlA and putative RmlA protein sequences, available in the NCBI data
base, was carried out using Multalign (34). The highly conserved
six-amino acid stretch LGDNI(F/Y) was used to design the degenerate
probe 5'-YTI GGI GAY AAY ATH TT-3' (where I is inosine, H is A/C/T, and
Y is C/T). This oligonucleotide was 3' end-labeled with digoxigenin,
and Southern hybridization experiments of completely digested
chromosomal DNA of A. thermoaerophilus DSM 10155 yielded
specific signals at 35 °C. A 4.5-kilobase pair BglII-fragment was isolated by preparative agarose gel
electrophoresis and cloned into the plasmid pBCKS, linearized with the
endonuclease BamHI. The corresponding construct pRML was sequenced.
Plasmid Construction--
Oligonucleotide primer sequences are
given in Table I.
Primers for the amplification of DNA fragments containing the
gmhA (primers GMHAA1 and GMHAA2), the kinase
(primers GMHBA1 and GMHBA2), or the pyrophosphorylase (primers GMHDA1
and GMHDA2) gene were designed with attB1 or attB2 sites for the
insertion into the GATEWAY donor vector pDONR201 by homologous
recombination. The PCR products were cloned into pDONR201, and the
resulting plasmids gGMHA1, gGMHB1, and gGMHD1 were used to transfer the gene sequences into pDEST15 (GST fusion) or pDEST17 (histidine fusion)
via homologous recombination. The corresponding plasmids gGMHA3
(histidine fusion), gGMHB3 (histidine fusion), and gGMHD2 (GST fusion)
were used for overexpression of the fusion proteins in E. coli BL21-SI.
Primers GMHC1F and GMHC2R were used for the amplification of the
phosphatase gene. The PCR product was digested with BglII and EcoRI and ligated into linearized and dephosphorylated
vector pBADB. The resulting plasmid pGMHC1 was used for
histidine-tagged fusion protein overexpression in E. coli LMG194.
Enzyme Purification--
E. coli BL21-SI cells
harboring the histidine-fusion plasmid gGMHA3 were grown at 37 °C to
an optical density at 600 nm of 0.5 in a 1-liter culture volume and
expression was induced for 150 min. E. coli BL21-SI carrying
gGMHB3 was grown at 30 °C to an optical density at 600 nm of 0.5 in
a 2-liter culture volume, and expression was induced overnight.
E. coli LMG194 harboring pgmhC1 was grown at 37 °C to an
optical density at 600 nm of 0.5 in a 1-liter culture volume, and
expression was induced for 3 h. Finally, E. coli
BL21-SI cells carrying the GST-fusion plasmid gGMHD2 were grown at
30 °C to an optical density at 600 nm of 0.6 in a 2-liter culture
volume, and expression was done overnight. Production of fusion protein
was induced by the addition of NaCl to a final concentration of 0.3 M for E. coli BL21-SI, or with L-arabinose to a final concentration of 0.2% for E. coli LMG194. The cells were disrupted by ultrasonication on ice,
cell debris were removed by centrifugation at 31,000 × g, and membrane fractions were collected as a pellet by
ultracentrifugation at 331,000 × g. All protein
purifications were done on an FPLC system of Amersham Pharmacia Biotech
using appropriate amounts of supernatant from the ultracentrifugation
step. Purifications using HiTrap Chelating and GSTrap columns were
performed as recommended by the manufacturer. For buffer exchange, a
HiPrep Desalting column was used. Anion exchange chromatography was
carried out in 20 mM Tris-HCl buffer, pH 7.7, using 1 M KCl as eluent. Dithiothreitol was added to a final
concentration of 1 mM during all purification steps, except when using HiTrap Chelating columns. Purification of GmhA, the putative
kinase, and the putative phosphatase was performed using affinity
chromatography on a HiPrep Chelating column. GmhB and GmhC were further
purified by anion exchange chromatography; the kinase eluted at 300 mM KCl, and the phosphatase eluted at 230 mM
KCl. The GST-tagged fusion protein of the putative pyrophosphorylase was purified using a GSTrap column. The enzymes were stored at 4 °C
or after stabilization with 50% glycerol at Enzyme Assays--
Ten nmol of D-sedoheptulose
7-phosphate or D- Enzymatic Synthesis of D,D-Heptose
Phosphates and GDP-D- NMR Spectroscopy--
Samples were dissolved in 99.95%
D2O (0.5 ml). Spectra were recorded at 300 K at 300.13 MHz
for 1H and at 75.47 MHz for 13C with a Bruker
AVANCE 300 spectrometer equipped with a 5-mm quadruple nuclear inverse
probehead with z-gradients. Data acquisition and processing
were performed with the standard XWINNMR software (Bruker). 1H spectra were referenced to
2,2-dimethyl-2-silapentane-5-sulfonic acid ( Cloning of the Heptose Operon--
The genes coding for
enzymes involved in the biosynthesis of a nucleotide-activated sugar
precursor generally are clustered within the corresponding gene cluster
for a particular bacterial polysaccharide (38). For the genes in the
dTDP-L-rhamnose biosynthetic pathway, this proposal was
verified in Gram-negative as well as in Gram-positive organisms (39,
40).2 To clone the genes involved in S-layer glycoprotein
glycan biosynthesis of A. thermoaerophilus DSM 10155, a
strategy was applied using a degenerate probe derived from RmlA protein
sequences (Fig. 1). Characterization of
the rml genes will be published elsewhere.2 The
insert of the cloned construct pRML was sequenced and contained the rmlA gene, incomplete at the 3' end, and four additional
ORFs upstream of rmlA. One of these open reading frames
codes for a homologue of GmhA, the first enzyme involved in the
biosynthesis of nucleotide-activated heptose. The fourth ORF was
incomplete, and its 5' end was sequenced using a recently described
method (41), revealing two additional ORFs. Again, the 5' end of the most upstream gene was missing.
Sequence Analyses--
The physical map of the sequenced
6,652-base pair DNA fragment is depicted in Fig. 1. According to
sequence comparisons with other RmlA proteins, the enzyme lacks only
few amino acids on its carboxyl terminus. Upstream of the
rmlA gene is a region of 939 base pairs without any
predicted ORF coding for proteins larger than 80 amino acids. The first
significant ORF upstream of rmlA codes for a protein of 179 amino acids. A Blast search yielded hypothetical ORFs of Vibrio
cholerae, C. jejuni, E. coli, H. pylori, and H. influenzae, and the bifunctional HisB
protein of Buchnera aphidicola and E. coli,
exhibiting imidazole glycerol phosphate dehydratase and
histidinol-phosphate phosphatase activities (42, 43). An alignment of
the amino acid sequences encoded by these hypothetical ORFs was
performed using Multalign (Ref. 34; see Fig.
2). The six hypothetical proteins showed
remarkable homology, and thus, are expected to carry out the same
catalytic reaction (Table II). The next
ORF of the cloned fragment encodes a protein with similarities to
mannose 1-phosphate guanosyltransferases as well as to other
pyrophosphorylases transferring thymidine, adenine, cytidine, or
uridine nucleotides to sugar phosphates. The third gene upstream of
rmlA encodes a homologue of GmhA, the sedoheptulose
7-phosphate isomerase, catalyzing the conversion of
D-sedoheptulose 7-phosphate to
D,D-heptose 7-phosphate. Upstream of
gmhA lies an ORF coding for a protein similar to
hypothetical proteins of C. jejuni, Mycobacterium
tuberculosis, and Thermoplasma acidophilum and to
galactokinases from several organisms. The remaining two ORFs both show
weak similarities to glycosyl transferases. In summary, the 5'-part of
the sequenced fragment contains genes coding for GmhA, a putative sugar
kinase, a putative phosphatase, a hypothetical pyrophosphorylase, and
two putative glycosyl transferases. Four of these proteins were
hypothesized to function in the biosynthesis of nucleotide-activated
heptose (Fig. 3). In the predicted
pathway, accommodating the putative activities of the gene products,
GmhA converts D-sedoheptulose 7-phosphate to an anomeric
mixture of D,D-heptose 7-phosphate. Instead of
the originally proposed mutase step to get
D,D-heptose 1-phosphate from
D,D-heptose 7-phosphate (19), two enzymes may
actually catalyze this conversion. The putative kinase adds a phosphate
group at the C-1 hydroxyl group of D,D-heptose
7-phosphate to form D,D-heptose
1,7-bisphosphate, and the putative phosphatase removes the phosphate
group at the C-7 position to yield D,D-heptose
1-phosphate. Finally, the putative pyrophosphorylase transfers a
nucleotide to the heptose unit to give
NDP-D,D-heptose. To test this hypothesis, we
decided to overexpress the corresponding proteins in order to perform
functional studies.
Overexpression of the Heptose Biosynthesis
Genes--
Overexpression of these genes from a Gram-positive organism
in E. coli proved to be a difficult task. Codon usage
differs remarkably in these two organisms. Different expression systems had to be used to finally achieve overexpression of all four genes. GmhA expression could be performed using pBAD and GATEWAY systems. For
enzyme purification a histidine-tagged protein was expressed with the
GATEWAY vector gGMHA3, and purification was carried out using a HiTrap
Chelating column. Overnight expression of histidine-tagged putative
kinase using the GATEWAY vector gGMHB3 yielded sufficient amount of
protein for purification over HiTrap Chelating and MonoQ columns. The
putative phosphatase was expressed using the pBAD construct pgmhC1, and
the histidine-tagged fusion protein was purified like the kinase. Most
difficult was the overexpression of the last enzyme of the putative
pathway, namely the pyrophosphorylase. Expression of histine-tagged
fusion proteins using the pBAD as well as the GATEWAY systems occurred
at a negligible level. However, low amounts of GST-tagged fusion
protein could be purified after overnight expression with the GATEWAY
vector gGMHD2, using a GSTrap column. The molecular masses of the
denatured proteins were in good agreement with the calculated masses,
as judged by SDS-polyacrylamide gel electrophoresis analysis (GmhA, 25 kDa; putative kinase, 41.6 kDa; putative phosphatase, 25.2 kDa;
putative pyrophosphorylase, 55.2 kDa; Fig.
4). The purified proteins were used for
functional characterization of the biosynthetic pathway of
nucleotide-activated D,D-heptose.
Synthesis of D,D-Heptose 1-Phosphate from
D-Sedoheptulose 7-Phosphate--
The conversion of
D-sedoheptulose 7-phosphate to
D,D-heptose intermediate products was analyzed
by phosphate anion exchange chromatography on a CarboPac PA-1 column
combined with NMR experiments. GmhA converted
D-sedoheptulose 7-phosphate to an anomeric mixture of
D,D-heptose 7-phosphate in an equilibrium
reaction, where ~20% of D-sedoheptulose 7-phosphate were
isomerized to D,D-heptose 7-phosphate (Fig.
5A). The retention time of
D,D-heptose 7-phosphate (24.5 min) resembled
that of D-glucose 6-phosphate (22.8 min). This first
intermediate product was hydrolyzed with trifluoroacetic acid, and
monosaccharide analysis on a CarboPac PA-1 column identified the sugar
as D,D-heptose (data not shown). Further
characterization was performed using NMR analysis. The
1H-NMR spectrum of D,D-heptose
7-phosphate indicated the presence of two anomers, and, due to the
small amount of material, the spectrum could not be fully assigned.
However, measurement of a H, P-correlated spectrum indicated the
occurrence of a phosphomonoester 31P signal at Activation of D- In this report we have elucidated for the first time
the overall pathway for biosynthesis of a nucleotide-activated
D- Overexpression of the heptose biosynthesis genes was
difficult due to differing codon usage in E. coli and the
Gram-positive bacterium used in this study. The use of the special
expression host E. coli BL21-CodonPlus-RIL, which harbors a
plasmid carrying extra copies of rare tRNA genes for arginine (codons
AGA and AGG), isoleucine (codon AUA), and leucine (codon CUA), did not
enhance expression of the genes. Thus, codons for other amino acids
seem to be the reason for the low expression levels. Leucine UUA,
valine GUA, threonine ACA, arginine CGA, and glycine GGA are used at a
3-fold higher rate in the A. thermoaerophilus DSM 10155 heptose genes in comparison to the E. coli K-12 genome.
However, the use of different expression systems eventually led to
successful overexpression of all four heptose genes. Problems in
overexpressing both the kinase and the pyrophosphorylase could only be
overcome by overnight expression, although even under these conditions
expression of the latter enzyme was still at a very low level.
The four steps of the reaction cascade synthesizing
GDP-D- As described before, neither an enzyme catalyzing the mutase step
nor an alternative monofunctional phosphatase has been reported, so
far. A BLAST search using the A. thermoaerophilus
phosphatase yielded putative ORFs from V. cholerae N16961,
C. jejuni NCTC 11168, E. coli K-12, H. pylori 26695 (and also J99), and H. influenzae Rd.
Accession numbers of putative heptose biosynthesis genes of the
completely sequenced bacterial strains are given in Table II. For
brevity of this work, only one of the sequenced strains of H. pylori (26695) and Neisseria meningitidis (MC58) was
taken into account, but the genes are found in the genomes of
additional strains. Judging from the high homology throughout the whole
protein sequence, all of these enzymes are expected to carry out the
same catalytic reaction (Fig. 2). For the conversion of the anomeric mixture of D,D-heptose 7-phosphate to
D,D-heptose 1-phosphate, it seems reasonable
that, in Gram-positive and Gram-negative organisms, either the -D-manno-heptose 1-phosphate was eventually activated with GTP by the pyrophosphorylase GmhD to yield the final product
GDP-D-glycero-
-D-manno-heptose. The intermediate and end products were analyzed by high performance liquid chromatography. Nuclear magnetic resonance spectroscopy was used
to confirm the structure of these substances. This is the first report
of the biosynthesis of
GDP-D-glycero-
-D-manno-heptose in Gram-positive organisms. In addition, we propose a pathway for
biosynthesis of the nucleotide-activated form of
L-glycero-D-manno-heptose.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-L-rhamnose and D-
-D-heptose
units (3). So far, this is the only Gram-positive bacterium where
D,D-heptose has been described as constituent of a cellular component. The principal architecture of S-layer glycoproteins resembles that of the LPS of Gram-negative bacteria (4).
Both glycoconjugates exhibit a tripartite structural organization, where, in general, conserved core regions connect a glycan chain, composed of identical repeating units, either with the S-layer polypeptide or the lipid A of LPS. It has been proposed that comparable pathways are used for the biosynthesis of these similar glycoconjugates (2). Recently we were able to verify this proposal by investigation of
the biosynthesis of nucleotide-activated D-rhamnose (5) and
L-rhamnose2 in
A. thermoaerophilus strains L420-91T and DSM
10155, respectively.
:K20
) (9), and Helicobacter
pylori AF1 and 007 (10) have been shown to contain both types of
heptose in LPS: L,D-heptose in the inner core
and D,D-heptose in the outer core. In
Yersinia enterocolitica Ye75R, both
D,D-heptose and
L,D-heptose are part of the inner core (11). It
must be noted, however, that only part of the existing literature on
D,D-heptose and
L,D-heptose is listed above, and coverage is
confined to those references where complete structural studies have
been performed. L-Rhamnose, the second sugar of the
repeating unit of the S-layer glycan of A. thermoaerophilus
DSM 10155 (3), is usually a constituent of the outer core region as
well as of the O-antigen of LPS (6, 12).
-D-heptose as substrate, and the
D-
-D isomer is also accepted, albeit with
10-fold reduced efficiency (23).
-D-heptose in Gram-positive
bacteria, and propose a general reaction pathway for the biosynthesis
of activated L,D-heptose in Gram-negative bacteria. In addition, we present a modified nomenclature scheme for
genes involved in heptose biosynthesis.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-manno-heptose
1-phosphate and
D-glycero-
-D-manno-heptose 1-phosphate were kindly provided by A. Zamyatina (Universität für Bodenkultur Wien, Wien, Austria). Glutathione (reduced form) was obtained from Merck GmbH (Wien, Austria). GSTrap, HiTrap Chelating, HiPrep Desalting, MonoQ HR5/5, and Sephadex G-10 were purchased from
Amersham Pharmacia Biotech (Uppsala, Sweden). The GATEWAY system was
obtained from Life Technologies, Inc. (Wien, Austria), and the pBAD
system was from Invitrogen (Groningen, The Netherlands). Ultrafree-MC
10000 ultrafiltration cartridges were purchased from Millipore
(Bedford, MA), and pBCKS was obtained from Stratagene (La Jolla, CA).
and E. coli TG1 were used for plasmid propagation. For enzyme
overexpression E. coli BL21-SI, E. coli
BL21-CodonPlus(DE3)-RIL, and E. coli LMG194 were used.
Protein overexpression was induced by the addition of NaCl,
isopropyl-1-thio-
-D-galactopyranoside, or
L-arabinose, respectively. Media were supplemented with
ampicillin, kanamycin, or chloramphenicol at concentrations of 100, 50, and 34 µg/ml, respectively, when required. Cells were grown at
30 °C or at 37 °C, with or without agitation.
Bacterial strains, plasmids, and oligonucleotides used in this study
20 °C. The purity of
the enzymes was checked by SDS-polyacrylamide gel electrophoresis analysis (4% stacking gel and 12% separating gel).
-D-heptose 1-phosphate were
used for enzyme assays. Fifty nmol of
-D-glucose 1,6-bisphosphate was used in a negative control assay to test the
specificity of D,D-heptose 1,7-bisphosphate
phosphatase. The assay buffer TH8 contained 20 mM Tris-HCl,
pH 8.0, and 10 mM MgCl2. Appropriate amounts of
enzymes were added, and, after 45 min of incubation at 37 °C, the
samples were analyzed by HPAEC.
-D-heptose--
One
µmol of D-sedoheptulose 7-phosphate was incubated with
appropriate amounts of GmhA in TH8 buffer at 37 °C until equilibrium was reached. The enzyme was removed by ultrafiltration using
Ultrafree-MC 10000 ultrafiltration cartridges.
D,D-Heptose 7-phosphate was separated from
D-sedoheptulose 7-phosphate by preparative HPAEC on a
CarboPac PA-1. The sample was desalted using a Carbohydrate Membrane
Desalter (CMD; Dionex), lyophilized, and investigated by NMR
spectroscopy. D,D-Heptose 7-phosphate, purified
in the first reaction step (~150 nmol), was incubated with 250 nmol
ATP and the kinase in 20 mM triethylammonium bicarbonate
buffer (pH 8.5; 10 mM MgCl2) at 37 °C. After
removal of the enzyme, the pH was adjusted to 3.0 by addition of
Dowex-50 ion exchange resin, the sample was lyophilized, and
D-
-D-heptose 1,7-bisphosphate was analyzed
by NMR spectroscopy. Three hundred nmol of D-sedoheptulose 7-phosphate were converted to D-
-D-heptose
1-phosphate, using appropriate amounts of GmhA, the kinase, the
phosphatase, and 600 nmol of ATP in TH8 buffer at 37 °C. The enzyme
mix was removed by ultrafiltration, and
D-
-D-heptose 1-phosphate was purified by
preparative HPAEC. The desalted (CMD) and lyophilized sample was
analyzed by NMR spectroscopy. Finally, 2 µmol of
D-
-D-heptose 1-phosphate were incubated with
the pyrophosphorylase and 3 µmol of GTP in TH8 buffer at 37 °C.
Inorganic pyrophosphatase was used to push the equilibrium reaction
toward GDP-D-
-D-heptose. The enzyme was
removed, and GDP-D-
-D-heptose was purified
on a semipreparative CarboPac PA-1 column. The sample was desalted
using Sephadex G-10 and lyophilized. NMR analysis was performed to
elucidate the structure of the final reaction product.
= 0),
13C spectra were referenced externally to 1,4-dioxane
(
= 67.40), and 31P spectra were referenced
externally to H3PO4 (
= 0). HMQC-, 1H,31P HMQC-, and
1H,31P heteronuclear single quantum
coherence-COSY spectra were recorded in the phase-sensitive mode
using time proportional phase incrementation and pulsed field gradients
for coherence selection (36, 37). Spectra resulted from a 256 × 4096 data matrix, with 1800 or 800 scans/t1 value, respectively.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
Physical map of the
GDP-D- -D-heptose
operon of A. thermoaerophilus DSM 10155. The
arrows indicate the genes and the hypothetical ORFs on the
sequence. gmhA, gmhB, gmhC, and
gmhD encode sedoheptulose 7-phosphate isomerase,
D,D-heptose 7-phosphate kinase,
D,D-heptose 1,7-bisphosphate phosphatase, and
D,D-heptose 1-phosphate guanosyltransferase,
respectively. rmlA encodes glucose 1-phosphate
thymidyltransferase, part of the pathway for
dTDP-L-rhamnose biosynthesis.
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Fig. 2.
Multiple sequence alignment of putative
phosphatase sequences. A. t., A. thermoaerophilus DSM 10155; C. j., C. jejuni
NCTC 11168; H. p., H. pylori 26695; H. i., H. influenzae Rd; E. c., E. coli K-12;
V. c., V. cholerae N16961.
Proteins putatively involved in the biosynthesis of
nucleotide-activated heptose
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Fig. 3.
Schematic representation of the biosynthesis
pathway of
GDP-D- -D-heptose.
A, sedoheptulose 7-phosphate isomerase GmhA; B,
D,D-heptose 7-phosphate kinase GmhB;
C, D,D-heptose 1,7-bisphosphate
phosphatase GmhC; D, D,D-heptose
1-phosphate guanosyltransferase GmhD.
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Fig. 4.
SDS-polyacrylamide gel
electrophoresis analysis of the purified enzymes involved in the
biosynthesis of
GDP-D- -D-heptose.
A, enzymes catalyzing the formation of
D-
-D-heptose 1-phosphate from sedoheptulose
7-phosphate. Lane 1, molecular mass standards, myosin (200 kDa),
-galactosidase (116.3 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66.3 kDa), glutamic dehydrogenase (55.4 kDa), lactate dehydrogenase (36.5 kDa), carbonic anhydrase (31 kDa),
trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa); lane
2, isomerase GmhA; lane 3, kinase GmhB; lane
4, phosphatase GmhC. B, enzyme activating
D-
-D-heptose 1-phosphate to form
GDP-D-
-D-heptose. Lane 1, see
A; lane 5, pyrophosphorylase GmhD.
3.30, which was coupled to a proton signal at
3.96. In addition, HMQC
correlation indicated the connectivity of this proton to a
13C signal at
65.5 and another proton at
4.10. Unambiguous evidence for the phosphate substitution at the terminal
hydroxymethyl group was obtained from the downfield shift of the C-7
signal and the upfield-shifted signal of the neighboring C-6 signal at
71.8. The connectivity of H- and C-6 to the phosphate-substituted
geminal CH2O protons was established from an H,P-correlated
COSY experiment. These data are in full agreement with similar effects
observed in the spectra of
L-glycero-D-manno-heptopyranose
7-phosphate (44). To investigate the next reaction step,
D-sedoheptulose 7-phosphate was incubated with GmhA, the
putative sugar kinase, and ATP as phosphate donor. In HPAEC analysis
the D-sedoheptulose 7-phosphate peak decreased,
D,D-heptose 7-phosphate disappeared, and a new
peak was detected, displaying a retention time of 31.2 min (Fig.
5B). Again, a peak occurred at a similar retention time compared with the corresponding glucose derivative, namely
-D-glucose 1,6-bisphosphate (31.6 min). The signal of
-D-glucose 1,6-bisphosphate was very weak and gave rise
to a very small peak in the chromatogram. As free hydroxyl groups are
electrochemically detected in Dionex-HPAEC, signal height decreases
with decreasing number of OH-groups. NMR analysis was used to elucidate
the structure of D,D-heptose 1,7-bisphosphate. For the NMR experiments, D,D-heptose
1,7-bisphosphate was synthesized from purified
D,D-heptose 7-phosphate. The H,P-correlated
spectrum of the D-
-D-heptose
1,7-bisphosphate displayed a phosphomonoester 31P signal at
1.22 coupled to the H-7 proton signals at
3.98 and 3.84, respectively. An additional 31P signal at
1.44 was
coupled to the anomeric proton at
5.39, thereby proving the
presence of the anomeric phosphate substituent. A mixture of
D-sedoheptulose 7-phosphate and ATP was also incubated with
only the kinase. The peak pattern did not change, which indicates the
specificity of the enzyme. The third step of the hypothetical reaction
sequence was supposed to be the removal of the phosphate on the C-7
position. This was investigated in a reaction cascade using the three
enzymes GmhA, the putative kinase, and the putative phosphatase.
D-Sedoheptulose 7-phosphate was incubated with the enzyme
mix and ATP. The D-sedoheptulose 7-phosphate peak decreased substantially, and the peaks corresponding to
D,D-heptose 7-phosphate and
D,D-heptose 1,7-bisphosphate disappeared
completely. A new peak was detected at 14.2 min, which is close to that
of
-D-glucose 1-phosphate (11.3 min; Fig.
5C). From the comparison with chemically synthesized
standards of D-
-D-heptose 1-phosphate (14.1 min) and D-
-D-heptose 1-phosphate (13.2 min)
(23), it was possible to identify the anomeric configuration of the
third intermediate product to be the
-anomer (Fig. 5C).
Final characterization of D-
-D-heptose
1-phosphate was performed by NMR analysis, which displayed a single
31P signal
2.53 coupled to the anomeric proton (
5.32). Furthermore, the spectral characteristics were in good agreement
with the synthetic compound as well as with published data (45). To
test the specificity of the phosphatase,
-D-glucose
1,6-bisphosphate was used as a substrate for this enzyme. Similar to
the kinase negative control, the peak pattern did not change. Whereas
the first reaction step, catalyzed by GmhA, is an equilibrium reaction,
the next two reactions appear to proceed quantitatively.
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Fig. 5.
HPAEC analysis of enzymatic synthesis of
D- -D-heptose
1-phosphate. A, D-sedoheptulose
7-phosphate, converted with the isomerase GmhA. B,
D-sedoheptulose 7-phosphate, converted with GmhA, the
kinase GmhB, and ATP. C, D-sedoheptulose
7-phosphate, converted with GmhA, GmhB, the phosphatase GmhC, and ATP.
Asterisk (*) marks retention time of synthetic
D-
-D-heptose 1-phosphate. D,
standard:
-D-glucose 1-phosphate, D-glucose
6-phosphate, D-sedoheptulose 7-phosphate,
-D-glucose 1,6-bisphosphate. Peaks with retention times
in the range of 30 and 35.5 min originate from adenosine phosphates and
dithiothreitol, respectively.
-D-Glucose
1,6-bisphosphate yields a very low signal and is therefore not visible
in the chromatogram.
-D-Heptose Using
GTP--
The final step in the biosynthetic pathway of
nucleotide-activated D,D-heptose is the
transfer of an NDP residue to D,D-heptose in
the C-1 position. Guanosine and adenosine had previously been described
as the activating nucleotide (20-22). To test which nucleotide is
actually used in the Gram-positive organism A. thermoaerophilus DSM 10155, D-
-D-heptose 1-phosphate was incubated with
the putative pyrophosphorylase and ATP or GTP, respectively. The
reaction mixtures were analyzed by HPAEC using a CarboPac PA-1 column.
No new peak appeared when ATP was used in the reaction (Fig.
6A). However, when the
guanosine nucleotide was used, the GTP peak decreased, and a new peak
with a retention time slower than GMP (13.3 min), but faster than GDP
(37 min), was detected (Fig. 6B). The retention time of 25.9 min was similar to that of GDP-D-mannose (28.4 min). The
reaction was also carried out with CTP, dTTP, and UTP, but no
additional peaks could be detected (data not shown). Thus, guanosine
diphosphate is the activating nucleotide of
D-
-D-heptose in the biosynthesis of the
S-layer glycoprotein glycan of A. thermoaerophilus DSM
10155. The end product of the pathway was fully characterized using
NMR analysis. The structure of the sugar nucleoside diphosphate, GDP-D-glycero-
-D-manno-heptopyranose,
was unambiguously confirmed by the NMR data and by comparison with both
anomers of synthetic ADP-D-glycero-D-manno-heptopyranose
(Fig. 7). The proton and carbon NMR
signals of position 8 of the guanine unit were observed at
8.09 and
138.6, respectively. In addition, the 1H NMR signals of
the
-D-ribofuranosyl residues were clearly separated from those of the heptose, which allowed the complete assignment of all
proton signals. Furthermore, HMQC experiments allowed a partial
detection of the connected carbons (Table
III). Both 31P signals of the
pyrophosphate group were detected and correlated to the anomeric proton
of the heptose and the H-5 protons of the ribose residue.
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Fig. 6.
HPAEC analysis of the activation of
D,D-heptose. A,
D- -D-heptose 1-phosphate, converted with the
pyrophosphorylase GmhD and ATP. B,
D-
-D-heptose 1-phosphate, converted with
GmhD and GTP. C, standard: NAD+, AMP, GMP, ADP,
GDP-D-mannose, GDP, ATP, and GTP.
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Fig. 7.
1H NMR analysis of
GDP-D- -D-heptose.
A, expansion plot of
ADP-D-
-D-heptose. B, expansion
plot of ADP-D-
-D-heptose. C,
expansion plot of GDP-D-
-D-heptose.
D, full spectrum of
GDP-D-
-D-heptose.
NMR data of GDP-D-glycero--D-manno-heptose
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-heptose from
D-sedoheptulose 7-phosphate. The heptose genes of A. thermoaerophilus DSM 10155 are clustered and could be identified
upstream of the rmlA gene. In this organism all genes
belonging to the dTDP-L-rhamnose operon are also
clustered.2 However, the genes for heptose biosynthesis are
not clustered in E. coli. In E. coli K-12, the
ADP-L,D-heptose epimerase gene rfaD,
the heptosyltransferase II gene rfaF, and the
heptosyltransferase I gene rfaC are part of a single operon,
whereas gmhA and ADP-heptose synthetase genes are located
elsewhere. The lack of an organized cluster of heptose biosynthesis
genes in prominent laboratory organisms like E. coli or
Salmonella enterica has hampered determination of the
pathway, as has the inability to identify a gene encoding an enzyme
catalyzing the mutase step, proposed in the biosynthetic pathway of
Eidels and Osborn (19). In addition, until now, genetic complementation
of heptose-deficient mutants did not identify an enzyme with
phosphatase homology. In the Gram-positive bacterium A. thermoaerophilus DSM 10155, however, four enzymes, the genes of
which are part of the same operon, are sufficient to synthesize GDP-D-
-D-heptose from
D-sedoheptulose 7-phosphate. This operon extends in the
upstream direction, but the whole cluster has not been sequenced yet.
Upstream of the four heptose genes are hypothetical ORFs, which encode
putative glycosyl transferases (Fig. 1), likely involved in S-layer
glycan synthesis.
-D-heptose from
D-sedoheptulose 7-phosphate in A. thermoaerophilus were unambiguously characterized by HPAEC
combined with NMR-spectroscopy. D-Sedoheptulose 7-phosphate
was converted to an anomeric mixture of
D,D-heptose 7-phosphate in an equilibrium
reaction. D,D-Heptose 7-phosphate was further
converted to D-
-D-heptose 1,7-bisphosphate in a quantitative reaction step using ATP as phosphate donor. H,P-correlation spectroscopy confirmed that there were two phosphate groups on one D-
-D-heptose unit: one
phosphorus atom being spin-coupled to the anomeric proton of C-1, and
the other coupled to the two protons of C-7. Thus, a mutase reaction
can be excluded, and synthesis of
D-
-D-heptose 1-phosphate from
D,D-heptose 7-phosphate proceeds via two
reaction steps involving a kinase and a phosphatase. The phosphatase
reaction was convincingly demonstrated in a three-step reaction
cascade, where D-
-D-heptose 1-phosphate was
isolated by preparative HPAEC, which was identical to authentic
synthetic material. The final product of the heptose biosynthetic
pathway is
GDP-D-glycero-
-D-manno-heptose.
- or
the
-anomer is taken up selectively by a phosphokinase (Fig.
8). The subsequent removal of the
7-phosphate group is expected to be performed by a phosphatase. Such
phosphatases can also be found in Pseudomonas aeruginosa
PAO1 and N. meningitidis MC58 and Z2491, however, with
lower homologies (Table II). To date, all heptose-containing bacteria
that have been completely sequenced contain a gene encoding a protein
homologous to the phosphatase GmhC of A. thermoaerophilus. M. tuberculosis H37Rv also has a gene homologous to this phosphatase
despite the fact that there is currently no report of
heptose-containing saccharides in this organism. Homologues of the
first three enzymes of the heptose pathway are part of a single operon,
whereas the last enzyme can also be found elsewhere on the chromosome
in this strain.
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Fig. 8.
Comparison of heptose biosynthetic
pathways. Left column, biosynthesis of
GDP-D- -D-heptose in A. thermoaerophilus DSM 10155. Right column,
putative pathway for biosynthesis of
ADP-L-
-D-heptose in Gram-negative bacteria.
For details, see "Discussion."
As far as currently known from Gram-negative bacteria (except N. meningitidis, see below), enzymes for heptose biosynthesis appear to be encoded by four genes, one of which encodes a bifunctional protein (26). This bifunctional enzyme putatively catalyzes two non-contiguous steps of the pathway, namely the addition of a phosphate residue to D,D-heptose 7-phosphate to give D,D-heptose 1,7-bisphosphate and the transfer of the nucleotide to D,D-heptose 1-phosphate to yield ADP-D,D-heptose. The four genes gmhA, the bifunctional putative kinase and pyrophosphorylase, the putative phosphatase, and ADP-L,D-heptose epimerase can be found on the chromosomes of C. jejuni NCTC 11168, E. coli K-12, H. influenzae Rd, H. pylori strains 26695 and J99, and P. aeruginosa PAO1 (Table II). In E. coli K-12, H. influenzae Rd, and P. aeruginosa PAO1, these genes are scattered on the chromosome, whereas in C. jejuni NCTC 11168 and H. pylori 26695 and J99, the genes are part of a single operon. N. meningitidis strains MC58 and Z2491 each possess five heptose biosynthesis genes scattered all over their chromosomes. In this organism the bifunctional putative kinase and pyrophosphorylase is split into two separate enzymes. V. cholerae N16961 possesses gmhA and an unlinked phosphatase gene, but lacks homologues of the kinase, the guanosyltransferase, the bifunctional putative kinase/pyrophosphorylase, and the epimerase. C. jejuni NCTC 11168 seems to carry two sets of heptose biosynthesis genes on its chromosome. They are organized in two operons: genes homologous to gmhA, ADP-heptose synthetase, ADP-L,D-heptose epimerase, and a putative phosphatase are part of the same operon, which also contains glycosyl transferases. A second operon contains a possible sugar-phosphate nucleotidyltransferase, an additional gmhA gene, and a putative sugar kinase as well as possible glycosyl transferases, but lacks a phosphatase homologue. However, it is not known whether C. jejuni NCTC 11168 contains both D,D-heptose and L,D-heptose as cellular constituents. In the LPS core of this strain, only the L,D-form of heptose has been reported (46). Additional glycoconjugates are known to occur in the cell envelope of this organism (47). Although there was no indication for the presence of heptose in these glycoconjugates, two separate sets of genes would potentially facilitate for incorporation of D,D-heptose or L,D-heptose into the corresponding polysaccharide structure.
The biosynthetic pathway of heptose seems to diverge during
the kinase step (Fig. 8). The kinase GmhB from A. thermoaerophilus catalyzes the formation of
D--D-heptose 1,7-bisphosphate. Since homologies from GmhB of A. thermoaerophilus to the kinase
part of ADP-heptose synthetase from different Gram-negative bacteria are low, in the case of ADP-heptose biosynthesis this intermediate might be D-
-D-heptose 1,7-bisphosphate. For
the phosphatase step, which does not involve the anomeric phosphate
group, the homology to the corresponding genes is high (Fig. 2). The
D,D-heptose 1-phosphate guanosyltransferase
accepts D-
-D-heptose 1-phosphate, whereas the D,D-heptose 1-phosphate adenosyltransferase
would accept the putative D-
-D-heptose
1-phosphate.
Recently, a general nomenclature for bacterial
polysaccharide synthesis has been introduced (48). The three-letter
gene name gmh (glycero-manno-heptose) is used for
the heptose biosynthesis genes. As we have shown in A. thermoaerophilus, there are four steps to get GDP-activated
D,D-heptose from D-sedoheptulose
7-phosphate. Thus, the suffixes A-D are sufficient to describe this
pathway (Fig. 8). In Gram-negative bacteria, however, suffixes A-E
would be required to identify the five steps including the
epimerization step to make L,D-heptose.
gmhA has been assigned to sedoheptulose 7-phosphate
isomerase, and gmhD has been proposed for the
ADP-L,D-heptose epimerase gene. Since the
latter enzyme catalyzes the fifth step in the heptose pathway of
Gram-negative bacteria, we suggest that this gene be named gmhE.
gmhB with subscript or
might be assigned to the gene
encoding D,D-heptose 7-phosphate kinase, and
the proposed name for the gene encoding the
D,D-heptose 1,7-bisphosphate phosphatase should
be gmhC. Finally, gmhD with the subscript NTP
(e.g. gmhDATP or
gmhDGTP) might be assigned to the gene encoding
the enzyme carrying out the D,D-heptose
1-phosphate nucleosyltransferase step. As described above, a
bifunctional enzyme, currently named ADP-heptose synthetase, putatively
catalyzes the addition of a phosphate group to
D,D-heptose 7-phosphate and the activation of
D,D-heptose 1-phosphate. In our scheme these
two reactions are catalyzed by gmhB and gmhD,
respectively. Therefore, gmhBD would be an appropriate name
for the gene encoding this bifunctional enzyme (Fig. 8).
In this study we have shown biosynthesis of
GDP-D--D-heptose in a Gram-positive
bacterium. To test our proposal for the heptose pathway, the enzymes
gmhBD and gmhC from E. coli are
currently under investigation. As there are no genetic tools presently
available for the Gram-positive bacterium A. thermoaerophilus, knock-out experiments with the
D,D-heptose 1,7-bisphosphate phosphatase gene
gmhC will be performed in E. coli K-12 to assess
its involvement in heptose biosynthesis.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. C. Whitfield (University of Guelph, Guelph, Ontario, Canada) for stimulating discussions and critical reading of the manuscript, Dr. A. Zamyatina for providing synthetic compounds and NMR spectra, and S. Zayni and A. Scheberl for excellent technical assistance.
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FOOTNOTES |
---|
* This work was supported by the Austrian Science Fund (projects P12966-MOB and P14209-MOB to P. M.).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) AF324836.
¶ To whom correspondence should be addressed. Tel.: 43-1-47654 (ext. 2202); Fax: 43-1-4789112; E-mail: pmessner@edv1.boku.ac.at.
Published, JBC Papers in Press, March 28, 2001, DOI 10.1074/jbc.M100378200
2 M. Graninger, B. Kneidinger, K. Bruno, and P. Messner, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: S-layer, surface layer; D, D-heptose, D-glycero-D-manno-heptose; LPS, lipopolysaccharide; L, D-heptose, L-glycero-D-manno-heptose; HPAEC, high-performance anion exchange chromatography; ORF, open reading frame; PCR, polymerase chain reaction; GST, glutathione S-transferase; HMQC, heteronuclear multiple quantum coherence.
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