Synthesis of the Carba-Analogs of the α-Pyranose and ß-Pyranose Forms of Sedoheptulose 7-Phosphate and Probing the Stereospecificity of Sedoheptulose 7-Phosphate Cyclases.

Sedoheptulose 7-phosphate (SH7P) cyclases are a subset of sugar phosphate cyclases that are known to catalyze the first committed step in many biosynthetic pathways in primary and secondary metabolism. Among them are 2-epi-5-epi-valiolone synthase (EEVS) and 2-epi-valiolone synthase (EVS), two closely related SH7P cyclases that catalyze the conversion of SH7P to 2-epi-5-epi-valiolone and 2-epi-valiolone, respectively. However, how these two homologous enzymes use a common substrate to produce stereochemically different products is unknown. Two competing hypotheses have been proposed for the stereospecificity of EEVS and EVS: (1) variation in aldol acceptor geometry during enzyme catalysis, and (2) preselection of the α-pyranose or ß-pyranose forms of the substrate by the enzymes. Yet, there is no direct evidence to support or rule out either of these hypotheses. Here we report the synthesis of the carba-analogs of the α-pyranose and ß-pyranose forms of SH7P and their use in probing the stereospecificity of ValA (EEVS from Streptomyces hygroscopicus subsp. jinggangensis) and Amir_2000 (EVS from Actinosynnema mirum DSM 43827). Kinetic studies of the enzymes in the presence of the synthetic compounds as well as docking studies of the enzymes with the α- and ß-pyranose forms of SH7P suggest that the inverted configuration of the products of EEVS and EVS is not due to the preselection of the different forms of the substrate by the enzymes.

Activation of Purine Biosynthesis Suppresses the Sensitivity of E. coli gmhA Mutant to Antibiotics.

Inactivation of enzymes responsible for biosynthesis of the cell wall component of ADP-glycero-manno-heptose causes the development of oxidative stress and sensitivity of bacteria to antibiotics of a hydrophobic nature. The metabolic precursor of ADP-heptose is sedoheptulose-7-phosphate (S7P), an intermediate of the non-oxidative branch of the pentose phosphate pathway (PPP), in which ribose-5-phosphate and NADPH are generated. Inactivation of the first stage of ADP-heptose synthesis (ΔgmhA) prevents the outflow of S7P from the PPP, and this mutant is characterized by a reduced biosynthesis of NADPH and of the Glu-Cys-Gly tripeptide, glutathione, molecules known to be involved in the resistance to oxidative stress. We found that the derepression of purine biosynthesis (∆purR) normalizes the metabolic equilibrium in PPP in ΔgmhA mutants, suppressing the negative effects of gmhA mutation likely via the over-expression of the glycine-serine pathway that is under the negative control of PurR and might be responsible for the enhanced synthesis of NADPH and glutathione. Consistently, the activity of the soxRS system, as well as the level of glutathionylation and oxidation of proteins, indicative of oxidative stress, were reduced in the double ΔgmhAΔpurR mutant compared to the ΔgmhA mutant.

Helicobacter pylori Modulates Heptose Metabolite Biosynthesis and Heptose-Dependent Innate Immune Host Cell Activation by Multiple Mechanisms.

Heptose metabolites including ADP-d-glycero-ß-d-manno-heptose (ADP-heptose) are involved in bacterial lipopolysaccharide and cell envelope biosynthesis. Recently, heptoses were also identified to have potent proinflammatory activity on human cells as novel microbe-associated molecular patterns. The gastric pathogenic bacterium Helicobacter pylori produces heptose metabolites, which it transports into human cells through its Cag type 4 secretion system. Using H. pylori as a model, we have addressed the question of how proinflammatory ADP-heptose biosynthesis can be regulated by bacteria. We have characterized the interstrain variability and regulation of heptose biosynthesis genes and the modulation of heptose metabolite production by H. pylori, which impact cell-autonomous proinflammatory human cell activation. HldE, a central enzyme of heptose metabolite biosynthesis, showed strong sequence variability between strains and was also variably expressed between strains. Amounts of gene transcripts in the hldE gene cluster displayed intrastrain and interstrain differences, were modulated by host cell contact and the presence of the cag pathogenicity island, and were affected by carbon starvation regulator A (CsrA). We reconstituted four steps of the H. pylori lipopolysaccharide (LPS) heptose biosynthetic pathway in vitro using recombinant purified GmhA, HldE, and GmhB proteins. On the basis of one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry, the structures of major reaction products were identified as ß-d-ADP-heptose and ß-heptose-1-monophosphate. A proinflammatory heptose-monophosphate variant was also identified for the first time as a novel cell-active product in H. pylori bacteria. Separate purified HldE subdomains and variant HldE allowed us to uncover additional strain variation in generating heptose metabolites. IMPORTANCE Bacterial heptose metabolites, intermediates of lipopolysaccharide (LPS) biosynthesis, are novel microbe-associated molecular patterns (MAMPs) that activate proinflammatory signaling. In the gastric pathogen Helicobacter pylori, heptoses are transferred into host cells by the Cag type IV secretion system, which is also involved in carcinogenesis. Little is known about how H. pylori, which is highly strain variable, regulates heptose biosynthesis and downstream host cell activation. We report here that the regulation of proinflammatory heptose production by H. pylori is strain specific. Heptose gene cluster activity is modulated by the presence of an active cag pathogenicity island (cagPAI), contact with human cells, and the carbon starvation regulator A. Reconstitution with purified biosynthesis enzymes and purified bacterial lysates allowed us to biochemically characterize heptose pathway products, identifying a heptose-monophosphate variant as a novel proinflammatory metabolite. These findings emphasize that the bacteria use heptose biosynthesis to fine-tune inflammation and also highlight opportunities to mine the heptose biosynthesis pathway as a potential therapeutic target against infection, inflammation, and cancer.

In vitro kinase assay reveals ADP-heptose-dependent ALPK1 autophosphorylation and altered kinase activity of disease-associated ALPK1 mutants.

Alpha-protein kinase 1 (ALPK1) is a pathogen recognition receptor that detects ADP-heptose (ADPH), a lipopolysaccharide biosynthesis intermediate, recently described as a pathogen-associated molecular pattern in Gram-negative bacteria. ADPH binding to ALPK1 activates its kinase domain and triggers TIFA phosphorylation on threonine 9. This leads to the assembly of large TIFA oligomers called TIFAsomes, activation of NF-κB and pro-inflammatory gene expression. Furthermore, mutations in ALPK1 are associated with inflammatory syndromes and cancers. While this kinase is of increasing medical interest, its activity in infectious or non-infectious diseases remains poorly characterized. Here, we use a non-radioactive ALPK1 in vitro kinase assay based on the use of ATPγS and protein thiophosphorylation. We confirm that ALPK1 phosphorylates TIFA T9 and show that T2, T12 and T19 are also weakly phosphorylated by ALPK1. Interestingly, we find that ALPK1 itself is phosphorylated in response to ADPH recognition during Shigella flexneri and Helicobacter pylori infection and that disease-associated ALPK1 mutants exhibit altered kinase activity. In particular, T237M and V1092A mutations associated with ROSAH syndrome and spiradenoma/spiradenocarcinoma respectively, exhibit enhanced ADPH-induced kinase activity and constitutive assembly of TIFAsomes. Altogether, this study provides new insights into the ADPH sensing pathway and disease-associated ALPK1 mutants.

The Inactivation of LPS Biosynthesis Genes in E. coli Cells Leads to Oxidative Stress.

Impaired lipopolysaccharide biosynthesis in Gram-negative bacteria results in the "deep rough" phenotype, which is characterized by increased sensitivity of cells to various hydrophobic compounds, including antibiotics novobiocin, actinomycin D, erythromycin, etc. The present study showed that E. coli mutants carrying deletions of the ADP-heptose biosynthesis genes became hypersensitive to a wide range of antibacterial drugs: DNA gyrase inhibitors, protein biosynthesis inhibitors (aminoglycosides, tetracycline), RNA polymerase inhibitors (rifampicin), and ß-lactams (carbenicillin). In addition, it was found that inactivation of the gmhA, hldE, rfaD, and waaC genes led to dramatic changes in the redox status of cells: a decrease in the pool of reducing NADPH and ATP equivalents, the concentration of intracellular cysteine, a change in thiol homeostasis, and a deficiency in the formation of hydrogen sulfide. In "deep rough" mutants, intensive formation of reactive oxygen species was observed, which, along with a lack of reducing agents, such as reactive sulfur species or NADPH, leads to oxidative stress and an increase in the number of dead cells in the population. Within the framework of modern ideas about the role of oxidative stress as a universal mechanism of the bactericidal action of antibiotics, inhibition of the enzymes of ADP-heptose biosynthesis is a promising direction for increasing the effectiveness of existing antibiotics and solving the problem of multidrug resistance.

Co-ordinated control of the ADP-heptose/ALPK1 signalling network by the E3 ligases TRAF6, TRAF2/c-IAP1 and LUBAC.

ADP-heptose activates the protein kinase ALPK1 triggering TIFA phosphorylation at Thr9, the recruitment of TRAF6 and the subsequent production of inflammatory mediators. Here, we demonstrate that ADP-heptose also stimulates the formation of Lys63- and Met1-linked ubiquitin chains to activate the TAK1 and canonical IKK complexes, respectively. We further show that the E3 ligases TRAF6 and c-IAP1 operate redundantly to generate the Lys63-linked ubiquitin chains required for pathway activation, which we demonstrate are attached to TRAF6, TRAF2 and c-IAP1, and that c-IAP1 is recruited to TIFA by TRAF2. ADP-heptose also induces activation of the kinase TBK1 by a TAK1-independent mechanism, which require TRAF2 and TRAF6. We establish that ALPK1 phosphorylates TIFA directly at Thr177 as well as Thr9 in vitro. Thr177 is located within the TRAF6-binding motif and its mutation to Asp prevents TRAF6 but not TRAF2 binding, indicating a role in restricting ADP-heptose signalling. We conclude that ADP-heptose signalling is controlled by the combined actions of TRAF2/c-IAP1 and TRAF6.

Kinetic Characterization and Computational Modeling of Escherichia coli Heptosyltransferase II: Exploring the Role of Protein Dynamics in Catalysis for GT-B Glycosyltransferase.

Glycosyltransferase (GT) enzymes promote the formation of glycosidic bonds between a sugar molecule and a diversity of substrates. Heptosyltransferase II (HepII) is a GT involved in the lipopolysaccharide (LPS) biosynthetic pathway that transfers the seven-carbon sugar (l-glycero-d-manno-heptose, Hep) onto a lipid-anchored glycopolymer (heptosylated Kdo2-Lipid A, Hep-Kdo2-Lipid A, or HLA). LPS plays a key role in Gram-negative bacterial sepsis, biofilm formation, and host colonization, and as such, LPS biosynthetic enzymes are targets for novel antimicrobial therapeutics. Three heptosyltransferases are involved in the inner-core LPS biosynthesis, with Escherichia coli HepII being the last to be quantitatively characterized in vivo. HepII shares modest sequence similarity with heptosyltransferase I (HepI) while maintaining a high degree of structural homology. Here, we report the first kinetic and biophysical characterization of HepII and demonstrate the properties of HepII that are shared with HepI, including sugar donor promiscuity and sugar acceptor-induced secondary structural changes, which results in significant thermal stabilization. HepII also has an increased catalytic efficiency and a significantly tighter binding affinity for both of its substrates compared to HepI. A structural model of the HepII ternary complex, refined by molecular dynamics simulations, was developed to probe the potentially important substrate-protein contacts. Ligand binding-induced changes in Trp fluorescence in HepII enabled the determination of substrate dissociation constants. Combined, these efforts meaningfully enhance our understanding of the heptosyltransferase family of enzymes and will aid in future efforts to design novel, potent, and specific inhibitors for this family of enzymes.

Reaction Mechanism and Three-Dimensional Structure of GDP-d-glycero-α-d-manno-heptose 4,6-Dehydratase from Campylobacter jejuni.

Campylobacter jejuni is a human pathogen and a leading cause of food poisoning in the United States and Europe. Surrounding the outside of the bacterium is a carbohydrate coat known as the capsular polysaccharide. Various strains of C. jejuni have different sequences of unusual sugars and an assortment of decorations. Many of the serotypes have heptoses with differing stereochemical arrangements at C2 through C6. One of the many common modifications is a 6-deoxy-heptose that is formed by dehydration of GDP-d-glycero-α-d-manno-heptose to GDP-6-deoxy-4-keto-d-lyxo-heptose via the action of the enzyme GDP-d-glycero-α-d-manno-heptose 4,6-dehydratase. Herein, we report the biochemical and structural characterization of this enzyme from C. jejuni 81-176 (serotype HS:23/36). The enzyme was purified to homogeneity, and its three-dimensional structure was determined to a resolution of 2.1 Å. Kinetic analyses suggest that the reaction mechanism proceeds through the formation of a 4-keto intermediate followed by the loss of water from C5/C6. Based on the three-dimensional structure, it is proposed that oxidation of C4 is assisted by proton transfer from the hydroxyl group to the phenolate of Tyr-159 and hydride transfer to the tightly bound NAD+ in the active site. Elimination of water at C5/C6 is most likely assisted by abstraction of the proton at C5 by Glu-136 and subsequent proton transfer to the hydroxyl at C6 via Ser-134 and Tyr-159. A bioinformatic analysis identified 19 additional 4,6-dehydratases from serotyped strains of C. jejuni that are 89-98% identical in the amino acid sequence, indicating that each of these strains should contain a 6-deoxy-heptose within their capsular polysaccharides.

A conservative distribution of tridomain NDP-heptose synthetases in actinobacteria.

Heptoses are important structural components of Gram-negative bacterium cell wall and participate in bacterial colonization, infection, and immune recognition. Current knowledge of NDP-heptose originating from D-sedoheptulose 7-phosphate in Grampositive bacterium remains limited. Here, in silico analysis suggested that the special tridomain NDP-heptose synthetases with isomerase, kinase, and nucleotidyltransferase activities are conservatively distributed in Actinobacteria class of Gram-positive bacterium. Enzymatical characterization of the tridomain proteins from different strains showed that they are involved in ADP-D-glycero-ß-D-manno-heptose biosynthesis despite the unexpected discovery of kinase activities deficient in some proteins. The presence of three types of NDP-heptose synthetases in Gram-positive bacterium suggests that it is also a rich source of heptoses and the heptose moieties may play important roles in vivo. Our work updates the understanding of NDP-heptose biosynthesis in Gram-positive bacterium and lays a solid foundation for further physiological function explorations.

ADP-heptose enables Helicobacter pylori to exploit macrophages as a survival niche by suppressing antigen-presenting HLA-II expression.

The persistence of Helicobacter pylori in the human gastric mucosa implies that the immune response fails to clear the infection. We found that H. pylori compromises the antigen presentation ability of macrophages, because of the decline of the presenting molecules HLA-II. Here, we reveal that the main bacterial factor responsible for this effect is ADP-heptose, an intermediate metabolite in the biosynthetic pathway of lipopolysaccharide (LPS) that elicits a pro-inflammatory response in gastric epithelial cells. In macrophages, it upregulates the expression of miR146b which, in turn, would downmodulate CIITA, the master regulator for HLA-II genes. Hence, H. pylori, utilizing ADP-heptose, exploits a specific arm of macrophage response to establish its survival niche in the face of the immune defense elicited in the gastric mucosa.

Biosynthesis of d-glycero-l-gluco-Heptose in the Capsular Polysaccharides of Campylobacter jejuni.

Campylobacter jejuni is the leading cause of food poisoning in the United States and Europe. The exterior cell surface of C. jejuni is coated with a capsular polysaccharide (CPS) that is essential for the maintenance and integrity of the bacterial cell wall and evasion of the host immune response. The identity and sequences of the monosaccharide components of the CPS are quite variable and dependent on the specific strain of C. jejuni. It is currently thought that the immediate precursor for the multiple variations found in the heptose moieties of the C. jejuni CPS is GDP-d-glycero-α-d-manno-heptose. In C. jejuni NCTC 11168, the heptose moiety is d-glycero-l-gluco-heptose. It has previously been shown that Cj1427 catalyzes the oxidation of GDP-d-glycero-α-d-manno-heptose to GDP-d-glycero-4-keto-α-d-lyxo-heptose using α-ketoglutarate as a cosubstrate. Cj1430 was now demonstrated to catalyze the double epimerization of this product at C3 and C5 to form GDP-d-glycero-4-keto-ß-l-xylo-heptose. Cj1428 subsequently catalyzes the stereospecific reduction of this GDP-linked heptose by NADPH to form GDP-d-glycero-ß-l-gluco-heptose. The three-dimensional crystal structure of Cj1430 was determined to a resolution of 1.85 Å in the presence of bound GDP-d-glycero-ß-l-gluco-heptose, a product analogue. The structure shows that it belongs to the cupin superfamily. The three-dimensional crystal structure of Cj1428 was solved in the presence of NADPH to a resolution of 1.50 Å. Its fold places it into the short-chain dehydrogenase/reductase superfamily. Typically, members in this family display a characteristic signature sequence of YXXXK, with the conserved tyrosine serving a key role in catalysis. In Cj1428, this residue is a phenylalanine.

Promoter-Assisted Stereoselective Synthesis of the 6-Deoxy-ß-d-manno-heptopyranose Oligosaccharides.

We report a promoter-assisted glycosidation approach for the stereoselective synthesis of the 6-deoxy-ß-d-manno-heptopyranose oligosaccharides. SphosAuNTf2-promoted glycosidation of 6-deoxy-d-manno-heptopyranosyl o-hexynylbenzoate with common alcohols afforded a range of 6-deoxy-d-manno-heptosides with good to excellent ß-selectivities. The counterion and the ligand of SPhosAuNTf2 were found to have a dramatic effect on the formation of the 1,2-cis-ß-linked 6-deoxy-d-manno-heptosides. This approach was effectively applied to the stereocontrolled synthesis of the 6-deoxy-ß-d-manno-heptopyranose oligosaccharides relevant to Burkholderia pseudomallei and Burkholderia mallei.

Heptose-containing bacterial natural products: structures, bioactivities, and biosyntheses.

Covering: up to 2020Glycosylated natural products hold great potential as drugs for the treatment of human and animal diseases. Heptoses, known as seven-carbon-chain-containing sugars, are a group of saccharides that are rarely observed in natural products. Based on the structures of the heptoses, the heptose-containing natural products can be divided into four groups, characterized by heptofuranose, highly-reduced heptopyranose, D-heptopyranose, and L-heptopyranose. Many of them possess remarkable biological properties, including antibacterial, antifungal, antitumor, and pain relief activities, thereby attracting great interest in biosynthesis and chemical synthesis studies to understand their construction mechanisms and structure-activity relationships. In this review, we summarize the structural properties, biological activities, and recent progress in the biosynthesis of bacterial natural products featuring seven-carbon-chain-containing sugars. The biosynthetic origins of the heptose moieties are emphasized.

Structure-function studies of the C3/C5 epimerases and C4 reductases of the Campylobacter jejuni capsular heptose modification pathways.

Many bacteria produce polysaccharide-based capsules that protect them from environmental insults and play a role in virulence, host invasion, and other functions. Understanding how the polysaccharide components are synthesized could provide new means to combat bacterial infections. We have previously characterized two pairs of homologous enzymes involved in the biosynthesis of capsular sugar precursors GDP-6-deoxy-D-altro-heptose and GDP-6-OMe-L-gluco-heptose in Campylobacter jejuni. However, the substrate specificity and mechanism of action of these enzymes-C3 and/or C5 epimerases DdahB and MlghB and C4 reductases DdahC and MlghC-are unknown. Here, we demonstrate that these enzymes are highly specific for heptose substrates, using mannose substrates inefficiently with the exception of MlghB. We show that DdahB and MlghB feature a jellyroll fold typical of cupins, which possess a range of activities including epimerizations, GDP occupying a similar position as in cupins. DdahC and MlghC contain a Rossman fold, a catalytic triad, and a small C-terminal domain typical of short-chain dehydratase reductase enzymes. Integrating structural information with site-directed mutagenesis allowed us to identify features unique to each enzyme and provide mechanistic insight. In the epimerases, mutagenesis of H67, D173, N121, Y134, and Y132 suggested the presence of alternative catalytic residues. We showed that the reductases could reduce GDP-4-keto-6-deoxy-mannulose without prior epimerization although DdahC preferred the pre-epimerized substrate and identified T110 and H180 as important for substrate specificity and catalytic efficacy. This information can be exploited to identify inhibitors for therapeutic applications or to tailor these enzymes to synthesize novel sugars useful as glycobiology tools.

Synthesis of l-glycero- and d-glycero-d-manno-Heptose Building Blocks for Stereoselective Assembly of the Lipopolysaccharide Core Trisaccharide of Vibrio parahemolyticus O2.

Synthesis of bacterial cell surface l-glycero-d-manno-heptose (l,d-Hep)- and d-glycero-d-manno-heptose (d,d-Hep)-containing higher carbon sugars is a challenging task. Here, we report a convenient and efficient approach for the synthesis of the l,d-Hep and d,d-Hep building blocks. Using l-lyxose and d-ribose as starting materials, this approach features diastereoselective Mukaiyama-type aldol reactions as the key steps. On the basis of the synthetic l,d-Hep and d,d-Hep building blocks, we achieved the first stereoselective synthesis of the unique α-l,d-Hep-(1→3)-α-d,d-Hep-(1→5)-α-Kdo core trisaccharide of the lipopolysaccharide of Vibrio parahemolyticus O2.

Nonhydrolyzable Heptose Bis- and Monophosphate Analogues Modulate Pro-inflammatory TIFA-NF-κB Signaling.

d-Glycero-d-manno-heptose-1ß,7-bisphosphate (HBP) and d-glycero-d-manno-heptose-1ß-phosphate (H1P) are bacterial metabolites that were recently shown to stimulate inflammatory responses in host cells through the activation of the TIFA-dependent NF-κB pathway. To better understand structure-based activity in relation to this process, a family of nonhydrolyzable phosphonate analogues of HBP and H1P was synthesized. The inflammation modulation by which these molecules induce the TIFA-NF-κB signal axis was evaluated in vivo at a low-nanomolar concentration (6 nM) and compared to that of the natural metabolites. Our data showed that three phosphonate analogues had similar stimulatory activity to HBP, whereas two phosphonates antagonized HBP-induced TIFA-NF-κB signaling. These results open new horizons for the design of pro-inflammatory and innate immune modulators that could be used as vaccine adjuvant.

Structure of a heptose-containing polysaccharide derived from Komagataeibacter europaeus NBRC 3261.

An O-antigen polysaccharide fraction (Ke-PS) was isolated from Komagataeibacter europaeus NBRC 3261. In addition to chemical analyses, its structure was characterized by 1D and 2D 1H as well as 13C NMR spectroscopy. The polysaccharide is composed of linear disaccharide repeating unit that consists of d-galactose and d-glycero-d-manno-heptose: →7)-α-d,d-Hepp-(1 â†’ 2)-α-d-Galp-(1 â†’ .

Functional Characterization of Cj1427, a Unique Ping-Pong Dehydrogenase Responsible for the Oxidation of GDP-d-glycero-α-d-manno-heptose in Campylobacter jejuni.

The capsular polysaccharides (CPS) of Campylobacter jejuni contain multiple heptose residues with variable stereochemical arrangements at C3-C6. The immediate precursor to all of these possible variations is currently believed to be GDP-d-glycero-α-d-manno-heptose. Oxidation of this substrate at C4 enables subsequent epimerization reactions at C3-C5 that can be coupled to the dehydration/reduction at C5/C6. However, the enzyme responsible for the critical oxidation of C4 from GDP-d-glycero-α-d-manno-heptose has remained elusive. The enzyme Cj1427 from C. jejuni NCTC 11168 was shown to catalyze the oxidation of GDP-d-glycero-α-d-manno-heptose to GDP-d-glycero-4-keto-α-d-lyxo-heptose in the presence of α-ketoglutarate using mass spectrometry and nuclear magnetic resonance spectroscopy. At pH 7.4, the apparent kcat is 0.6 s-1, with a value of kcat/Km of 1.0 × 104 M-1 s-1 for GDP-d-glycero-α-d-manno-heptose. α-Ketoglutarate is required to recycle the tightly bound NADH nucleotide in the active site of Cj1427, which does not dissociate from the enzyme during catalysis.

Structural Analysis of Cj1427, an Essential NAD-Dependent Dehydrogenase for the Biosynthesis of the Heptose Residues in the Capsular Polysaccharides of Campylobacter jejuni.

Many strains of Campylobacter jejuni display modified heptose residues in their capsular polysaccharides (CPS). The precursor heptose was previously shown to be GDP-d-glycero-α-d-manno-heptose, from which a variety of modifications of the sugar moiety have been observed. These modifications include the generation of 6-deoxy derivatives and alterations of the stereochemistry at C3-C6. Previous work has focused on the enzymes responsible for the generation of the 6-deoxy derivatives and those involved in altering the stereochemistry at C3 and C5. However, the generation of the 6-hydroxyl heptose residues remains uncertain due to the lack of a specific enzyme to catalyze the initial oxidation at C4 of GDP-d-glycero-α-d-manno-heptose. Here we reexamine the previously reported role of Cj1427, a dehydrogenase found in C. jejuni NTCC 11168 (HS:2). We show that Cj1427 is co-purified with bound NADH, thus hindering catalysis of oxidation reactions. However, addition of a co-substrate, α-ketoglutarate, converts the bound NADH to NAD+. In this form, Cj1427 catalyzes the oxidation of l-2-hydroxyglutarate back to α-ketoglutarate. The crystal structure of Cj1427 with bound GDP-d-glycero-α-d-manno-heptose shows that the NAD(H) cofactor is ideally positioned to catalyze the oxidation at C4 of the sugar substrate. Additionally, the overall fold of the Cj1427 subunit places it into the well-defined short-chain dehydrogenase/reductase superfamily. The observed quaternary structure of the tetrameric enzyme, however, is highly unusual for members of this superfamily.

d-Idose, d-Iduronic Acid, and d-Idonic Acid from d-Glucose via Seven-Carbon Sugars.

A practical synthesis of the very rare sugar d-idose and the stable building blocks for d-idose, d-iduronic, and d-idonic acids from ido-heptonic acid requires only isopropylidene protection, Shing silica gel-supported periodate cleavage of the C6-C7 bond of the heptonic acid, and selective reduction of C1 and/or C6. d-Idose is the most unstable of all the aldohexoses and a stable precursor which be stored and then converted under very mild conditions into d-idose is easily prepared.