Biosynthesis of Nucleotide-activated D-glycero-D-manno-Heptose*

Bernd KneidingerDagger , Michael GraningerDagger , Michael Puchberger§, Paul Kosma§, and Paul MessnerDagger

From the Dagger  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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha -D-manno-heptose 1-phosphate was eventually activated with GTP by the pyrophosphorylase GmhD to yield the final product GDP-D-glycero-alpha -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-alpha -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -L-rhamnose and D-beta -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.

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-: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).

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-beta -D-heptose as substrate, and the D-beta -D isomer is also accepted, albeit with 10-fold reduced efficiency (23).

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-alpha -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- ATP, GTP, D-sedoheptulose 7-phosphate, and dithiothreitol were obtained from Sigma (Sigma Aldrich Fluka GmbH, Wien, Austria). D-glycero-alpha -D-manno-heptose 1-phosphate and D-glycero-beta -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).

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 DH5alpha 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-beta -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.

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.

                              
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Table I
Bacterial strains, plasmids, and oligonucleotides used in this study

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 -20 °C. The purity of the enzymes was checked by SDS-polyacrylamide gel electrophoresis analysis (4% stacking gel and 12% separating gel).

Enzyme Assays-- Ten nmol of D-sedoheptulose 7-phosphate or D-alpha -D-heptose 1-phosphate were used for enzyme assays. Fifty nmol of alpha -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.

Enzymatic Synthesis of D,D-Heptose Phosphates and GDP-D-alpha -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-alpha -D-heptose 1,7-bisphosphate was analyzed by NMR spectroscopy. Three hundred nmol of D-sedoheptulose 7-phosphate were converted to D-alpha -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-alpha -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-alpha -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-alpha -D-heptose. The enzyme was removed, and GDP-D-alpha -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.

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 (delta  = 0), 13C spectra were referenced externally to 1,4-dioxane (delta  = 67.40), and 31P spectra were referenced externally to H3PO4 (delta  = 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
RESULTS
DISCUSSION
REFERENCES

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.


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Fig. 1.   Physical map of the GDP-D-alpha -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.

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.


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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.

                              
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Table II
Proteins putatively involved in the biosynthesis of nucleotide-activated heptose
Accession number is from the National Center for Biotechnology Information data base.


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Fig. 3.   Schematic representation of the biosynthesis pathway of GDP-D-alpha -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.

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.


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Fig. 4.   SDS-polyacrylamide gel electrophoresis analysis of the purified enzymes involved in the biosynthesis of GDP-D-alpha -D-heptose. A, enzymes catalyzing the formation of D-alpha -D-heptose 1-phosphate from sedoheptulose 7-phosphate. Lane 1, molecular mass standards, myosin (200 kDa), beta -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-alpha -D-heptose 1-phosphate to form GDP-D-alpha -D-heptose. Lane 1, see A; lane 5, pyrophosphorylase GmhD.

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 delta  3.30, which was coupled to a proton signal at delta  3.96. In addition, HMQC correlation indicated the connectivity of this proton to a 13C signal at delta  65.5 and another proton at delta  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 delta  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 alpha -D-glucose 1,6-bisphosphate (31.6 min). The signal of alpha -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-alpha -D-heptose 1,7-bisphosphate displayed a phosphomonoester 31P signal at delta  1.22 coupled to the H-7 proton signals at delta  3.98 and 3.84, respectively. An additional 31P signal at delta  -1.44 was coupled to the anomeric proton at delta  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 alpha -D-glucose 1-phosphate (11.3 min; Fig. 5C). From the comparison with chemically synthesized standards of D-alpha -D-heptose 1-phosphate (14.1 min) and D-beta -D-heptose 1-phosphate (13.2 min) (23), it was possible to identify the anomeric configuration of the third intermediate product to be the alpha -anomer (Fig. 5C). Final characterization of D-alpha -D-heptose 1-phosphate was performed by NMR analysis, which displayed a single 31P signal delta  2.53 coupled to the anomeric proton (delta  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, alpha -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-alpha -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-beta -D-heptose 1-phosphate. D, standard: alpha -D-glucose 1-phosphate, D-glucose 6-phosphate, D-sedoheptulose 7-phosphate, alpha -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. alpha -D-Glucose 1,6-bisphosphate yields a very low signal and is therefore not visible in the chromatogram.

Activation of D-alpha -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-alpha -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-alpha -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-alpha -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 delta  8.09 and delta  138.6, respectively. In addition, the 1H NMR signals of the beta -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-alpha -D-heptose 1-phosphate, converted with the pyrophosphorylase GmhD and ATP. B, D-alpha -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-alpha -D-heptose. A, expansion plot of ADP-D-beta -D-heptose. B, expansion plot of ADP-D-alpha -D-heptose. C, expansion plot of GDP-D-alpha -D-heptose. D, full spectrum of GDP-D-alpha -D-heptose.

                              
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Table III
NMR data of GDP-D-glycero-alpha -D-manno-heptose


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this report we have elucidated for the first time the overall pathway for biosynthesis of a nucleotide-activated D-alpha -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-alpha -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.

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-alpha -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-alpha -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-alpha -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-alpha -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-alpha -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-alpha -D-manno-heptose.

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 alpha - or the beta -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-alpha -D-heptose in A. thermoaerophilus DSM 10155. Right column, putative pathway for biosynthesis of ADP-L-beta -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-alpha -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-beta -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-alpha -D-heptose 1-phosphate, whereas the D,D-heptose 1-phosphate adenosyltransferase would accept the putative D-beta -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 alpha  or beta  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-alpha -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.

    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.

    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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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