Functional Characterization of Two PLP-Dependent Enzymes Involved in Capsular Polysaccharide Biosynthesis from Campylobacter jejuni.

Campylobacter jejuni is a Gram-negative, pathogenic bacterium that causes campylobacteriosis, a form of gastroenteritis. C. jejuni is the most frequent cause of food-borne illness in the world, surpassing Salmonella and E. coli. Coating the surface of C. jejuni is a layer of sugar molecules known as the capsular polysaccharide that, in C. jejuni NCTC 11168, is composed of a repeating unit of d-glycero-l-gluco-heptose, d-glucuronic acid, d-N-acetyl-galactosamine, and d-ribose. The d-glucuronic acid moiety is further amidated with either serinol or ethanolamine. It is unknown how these modifications are synthesized and attached to the polysaccharide. Here, we report the catalytic activities of two previously uncharacterized, pyridoxal phosphate (PLP)-dependent enzymes, Cj1436 and Cj1437, from C. jejuni NCTC 11168. Using a combination of mass spectrometry and nuclear magnetic resonance, we determined that Cj1436 catalyzes the decarboxylation of l-serine phosphate to ethanolamine phosphate. Cj1437 was shown to catalyze the transamination of dihydroxyacetone phosphate to (S)-serinol phosphate in the presence of l-glutamate. The probable routes to the ultimate formation of the glucuronamide substructures in the capsular polysaccharides of C. jejuni are discussed.

Helicobacter pylori GmhB enzyme involved in ADP-heptose biosynthesis pathway is essential for lipopolysaccharide biosynthesis and bacterial virulence.

Helicobacter pylori infection is linked to serious gastric-related diseases including gastric cancer. However, current therapies for treating H. pylori infection are challenged by the increased antibiotic resistance of H. pylori. Therefore, it is in an urgent need to identify novel targets for drug development against H. pylori infection. In this study, HP0860 gene from H. pylori predicted to encode a D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) involved in the synthesis of ADP-L-glycero-D-manno-heptose for the assembly of lipopolysaccharide (LPS) in the inner core region was cloned and characterized. We reported HP0860 protein is monomeric and functions as a phosphatase by converting D-glycero-D-manno-heptose-1,7-bisphosphate into D-glycero-D-manno-heptose-1-phosphate with a preference for the ß-anomer over the α-anomer of sugar phosphate substrates. Subsequently, a HP0860 knockout mutant and its complementary mutant were constructed and their phenotypic properties were examined. HP0860 knockout mutant contained both mature and immature forms of LPS and could still induce significant IL-8 secretion after gastric AGS cell infection, suggesting other enzymatic activities in HP0860 knockout mutant might be able to partially compensate for the loss of HP0860 activity. In addition, HP0860 knockout mutant was much more sensitive to antibiotic novobiocin, had decreased adherence abilities, and caused less classic hummingbird phenotype on the infected AGS cells, indicating H. pylori lacking HP0860 is less virulent. Furthermore, the disruption of HP0860 gene altered the sorting of cargo proteins into outer membrane vesicles (OMVs). The above findings confirm the importance of HP0860 in LPS core biosynthesis and shed light on therapeutic intervention against H. pylori infection.

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.

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.

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.

Biosynthesis of GDP-d-glycero-α-d-manno-heptose for the Capsular Polysaccharide of Campylobacter jejuni.

The capsular polysaccharide (CPS) structure of Campylobacter jejuni contributes to its robust fitness. Many strains contain heptose moieties in their CPS units. The precursor heptose is GDP-d-glycero-α-d-manno-heptose; modifications to the stereochemistry at C3-C6 as well as additions of methyl and phosphoramidate groups lend to the hypervariability of the C. jejuni CPS structures. Synthesis of GDP-d-glycero-α-d-manno-heptose has been described previously, but using enzymes from Aneurinibacillus thermoaerophilus DSM 10155. Here we describe the complete synthesis of GDP-d-glycero-α-d-manno-heptose using enzymes from C. jejuni NTCC 11168: Cj1152 and Cj1423-Cj1425. Our results yield kinetic parameters for these enzymes and outline a successful strategy for milligram-gram scale synthesis of GDP-d-glycero-α-d-manno-heptose. This achievement is critical for the characterization of other carbohydrate tailoring enzymes, which are expected to utilize GDP-d-glycero-α-d-manno-heptose for the biosynthesis of more complex carbohydrates in the CPS of C. jejuni.

Low-resolution SAXS and structural dynamics analysis on M. tuberculosis GmhB enzyme involved in GDP-heptose biosynthetic pathway.

The M. tuberculosis GmhB protein converts the d-glycero-α-d-manno-heptose 1,7-bisphosphate (GMB) intermediate into d-glycero-α-d-manno-heptose 1-phosphate by removing the phosphate group at the C-7 position. To understand the structure and substrate binding mechanism, the MtbGmhB was purified which elutes as monomer on gel filtration column. The small angle x-ray scattering analysis shows that MtbGmhB forms fully folded monomer with shape profile similar to its modeled structure. The circular dichroism analysis shows 38% α-helix, 15% ß-sheets and 47% random coil structures in MtbGmhB, similar to haloalkanoic acid dehalogenase (HAD) phosphohydrolase enzymes. The modeled MtbGmhB structure shows the catalytic site, which forms a concave, semicircular surface using the three loops around GMB substrate binding site. Dynamic simulation analysis on (i) Apo (ii) GMB bound (iii) GMB + Mg2+ bound (iv) Zn2+ +GMB + Mg2+ bound MtbGmhB structures show that Zn2+ as well as Mg2+ ions stabilize the loop conformation and trigger the changes in GMB substrate binding to active site of MtbGmhB. Upon demetallization, the large conformational changes occurred in ions binding loops, and leads to difference in GMB substrate binding to MtbGmhB. Our study provides information about structure and substrate binding of MtbGmhB, which may contribute in therapeutic development against M. tuberculosis.

ADP-heptose: A new innate immune modulator.

Lipopolysaccharide (LPS) is a well-known pathogen-associated molecular pattern (PAMP) produced by gram-negative bacteria. Previous studies showed that a key metabolic intermediate in LPS biosynthesis, d-glycero-ß-d-manno-heptose 1,7-bisphosphate (HBP), could activate the NF-κB pathway and trigger the innate immune responses. However, it was unclear whether HBP could be a novel PAMP and its pattern recognition receptor (PRR) is not fully understood. Very recently, the Shao group reported that another key metabolic intermediate in LPS biosynthesis, ADP-heptose, could be transported into mammalian cells and bind with ALPK1 (alpha-kinase 1), which leads to a series of strong immune responses. These findings broaden our understanding on bacterial metabolites as a new type of PAMP and these small molecules hold great potential to be applied in the development of novel immune modulators. This minireview focuses on the roles of ADP-heptose related metabolites in innate immunity.

Helicobacter pylori modulates host cell responses by CagT4SS-dependent translocation of an intermediate metabolite of LPS inner core heptose biosynthesis.

Highly virulent Helicobacter pylori cause proinflammatory signaling inducing the transcriptional activation and secretion of cytokines such as IL-8 in epithelial cells. Responsible in part for this signaling is the cag pathogenicity island (cagPAI) that codetermines the risk for pathological sequelae of an H. pylori infection such as gastric cancer. The Cag type IV secretion system (CagT4SS), encoded on the cagPAI, can translocate various molecules into cells, the effector protein CagA, peptidoglycan metabolites and DNA. Although these transported molecules are known to contribute to cellular responses to some extent, a major part of the cagPAI-induced signaling leading to IL-8 secretion remains unexplained. We report here that biosynthesis of heptose-1,7-bisphosphate (HBP), an important intermediate metabolite of LPS inner heptose core, contributes in a major way to the H. pylori cagPAI-dependent induction of proinflammatory signaling and IL-8 secretion in human epithelial cells. Mutants defective in the genes required for synthesis of HBP exhibited a more than 95% reduction of IL-8 induction and impaired CagT4SS-dependent cellular signaling. The loss of HBP biosynthesis did not abolish the ability to translocate CagA. The human cellular adaptor TIFA, which was described before to mediate HBP-dependent activity in other Gram-negative bacteria, was crucial in the cagPAI- and HBP pathway-induced responses by H. pylori in different cell types. The active metabolite was present in H. pylori lysates but not enriched in bacterial supernatants. These novel results advance our mechanistic understanding of H. pylori cagPAI-dependent signaling mediated by intracellular pattern recognition receptors. They will also allow to better dissect immunomodulatory activities by H. pylori and to improve the possibilities of intervention in cagPAI- and inflammation-driven cancerogenesis.

General assay for enzymes in the heptose biosynthesis pathways using electrospray ionization mass spectrometry.

The ADP-L-glycero-ß-D-manno-heptose and the GDP-6-deoxy-α-D-manno-heptose biosynthesis pathways play important roles in constructing lipopolysaccharide of Gram-negative bacteria. Blocking the pathways is lethal or increases antibiotic susceptibility to pathogens. Therefore, the enzymes involved in the pathways are novel antibiotic drug targets. Here, we designed an efficient method to assay the whole enzymes in the pathways using mass spectrometry and screened 148 compounds. One promising lead is (-)-nyasol targeting D-glycero-α-D-manno-heptose-1-phosphate guanylyltransferase (HddC) included in the GDP-6-deoxy-α-D-manno-heptose biosynthesis pathway from Burkholderia pseudomallei. The inhibitory activity of the lead compound against HddC has been confirmed by blocking the system transferring the guanosine monophosphate (GMP) moiety to α-D-glucose-1-phosphate. (-)-Nyasol exhibits the half maximal inhibitory concentration (IC50) value of 17.6 µM. A further study is going on using (-)-nyasol derivatives to find better leads with high affinity.

Functional and structural studies on the Neisseria gonorrhoeae GmhA, the first enzyme in the glycero-manno-heptose biosynthesis pathways, demonstrate a critical role in lipooligosaccharide synthesis and gonococcal viability.

Sedoheptulose-7-phosphate isomerase, GmhA, is the first enzyme in the biosynthesis of nucleotide-activated-glycero-manno-heptoses and an attractive, yet underexploited, target for development of broad-spectrum antibiotics. We demonstrated that GmhA homologs in Neisseria gonorrhoeae and N. meningitidis (hereafter called GmhAGC and GmhANM , respectively) were interchangeable proteins essential for lipooligosaccharide (LOS) synthesis, and their depletion had adverse effects on neisserial viability. In contrast, the Escherichia coli ortholog failed to complement GmhAGC depletion. Furthermore, we showed that GmhAGC is a cytoplasmic enzyme with induced expression at mid-logarithmic phase, upon iron deprivation and anaerobiosis, and conserved in contemporary gonococcal clinical isolates including the 2016 WHO reference strains. The untagged GmhAGC crystallized as a tetramer in the closed conformation with four zinc ions in the active site, supporting that this is most likely the catalytically active conformation of the enzyme. Finally, site-directed mutagenesis studies showed that the active site residues E65 and H183 were important for LOS synthesis but not for GmhAGC function in bacterial viability. Our studies bring insights into the importance and mechanism of action of GmhA and may ultimately facilitate targeting the enzyme with small molecule inhibitors.

Crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis in Escherichia coli.

The outer membrane of gram-negative bacteria is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. LPS is an endotoxin that elicits a strong immune response from humans, and its biosynthesis is in part regulated via degradation of LpxC (EC 3.5.1.108) and WaaA (EC 2.4.99.12/13) enzymes by the protease FtsH (EC 3.4.24.-). Because the synthetic pathways for both molecules are complex, in addition to being produced in strict ratios, we developed a computational model to interrogate the regulatory mechanisms involved. Our model findings indicate that the catalytic activity of LpxK (EC 2.7.1.130) appears to be dependent on the concentration of unsaturated fatty acids. This is biologically important because it assists in maintaining LPS/phospholipids homeostasis. Further crosstalk between the phospholipid and LPS biosynthetic pathways was revealed by experimental observations that LpxC is additionally regulated by an unidentified protease whose activity is independent of lipid A disaccharide concentration (the feedback source for FtsH-mediated LpxC regulation) but could be induced in vitro by palmitic acid. Further experimental analysis provided evidence on the rationale for WaaA regulation. Overexpression of waaA resulted in increased levels of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) sugar in membrane extracts, whereas Kdo and heptose levels were not elevated in LPS. This implies that uncontrolled production of WaaA does not increase the LPS production rate but rather reglycosylates lipid A precursors. Overall, the findings of this work provide previously unidentified insights into the complex biogenesis of the Escherichia coli outer membrane.

Biosynthesis of 4-aminoheptose 2-epimers, core structural components of the septacidins and spicamycins.

Septacidins and spicamycins are acylated 4-aminoheptosyl-ß-N-glycosides produced by Streptomyces fimbriatus and S. alanosinicus, respectively. Their structures are highly conserved, but differ in the stereochemistry of the 4-aminoheptosyl residues. The origin of this stereochemistry is unknown, but is presumably because of the difference in their biosynthetic pathways. We have synthesized the septacidin 4-aminoheptose to verify the difference between septacidin and spicamycin. Isotopic enrichment studies were undertaken using S. fimbriatus, and show that the septacidin heptose is derived from the pentose phosphate pathway. This indicates conserved pathways leading to the biosynthesis of 4-amino-4-deoxy-L-gluco-heptose or 4-amino-4-deoxy-L-manno-heptose.

Comparison of predicted epimerases and reductases of the Campylobacter jejuni D-altro- and L-gluco-heptose synthesis pathways.

Uniquely modified heptoses found in surface carbohydrates of bacterial pathogens are potential therapeutic targets against such pathogens. Our recent biochemical characterization of the GDP-6-deoxy-D-manno- and GDP-6-deoxy-D-altro-heptose biosynthesis pathways has provided the foundation for elucidation of the more complex L-gluco-heptose synthesis pathway of Campylobacter jejuni strain NCTC 11168. In this work we use GDP-4-keto,6-deoxy-D-lyxo-heptose as a surrogate substrate to characterize three enzymes predicted to be involved in this pathway: WcaGNCTC (also known as Cj1427), MlghB (Cj1430), and MlghC (Cj1428). We compare them with homologues involved in d-altro-heptose production: WcaG81176 (formerly WcaG), DdahB (Cjj1430), and DdahC (Cjj1427). We show that despite high levels of similarity, the enzymes have pathway-specific catalytic activities and substrate specificities. MlghB forms three products via C3 and C5 epimerization activities, whereas its DdahB homologue only had C3 epimerase activity along its cognate pathway. MlghC is specific for the double C3/C5 epimer generated by MlghB and produces L-gluco-heptose via stereospecific C4 reductase activity. In contrast, its homologue DdahC only uses the C3 epimer to yield d-altro-heptose via C4 reduction. Finally, we show that WcaGNCTC is not necessary for L-gluco-heptose synthesis and does not affect its production by MlghB and MlghC, in contrast to its homologue WcaG81176, that has regulatory activity on d-altro-heptose synthesis. These studies expand our fundamental understanding of heptose modification, provide new glycobiology tools to synthesize novel heptose derivatives with biomedical applications, and provide a foundation for the structure function analysis of these enzymes.

Complete 6-deoxy-D-altro-heptose biosynthesis pathway from Campylobacter jejuni: more complex than anticipated.

The Campylobacter jejuni capsule is important for colonization and virulence in various infection models. In most strains, the capsule includes a modified heptose whose biological role and biosynthetic pathway are unknown. To decipher the biosynthesis pathway for the 6-deoxy-D-altro-heptose of strain 81-176, we previously showed that the 4,6-dehydratase WcbK and the reductase WcaG generated GDP-6-deoxy-D-manno-heptose, but the C3 epimerase necessary to form GDP-6-deoxy-D-altro-heptose was not identified. Herein, we characterized the putative C3/C5 epimerase Cjj1430 and C3/C5 epimerase/C4 reductase Cjj1427 from the capsular cluster. We demonstrate that GDP-6-deoxy-D-altro-heptose biosynthesis is more complex than anticipated and requires the sequential action of WcbK, Cjj1430, and Cjj1427. We show that Cjj1430 serves as C3 epimerase devoid of C5 epimerization activity and that Cjj1427 has no epimerization activity and only serves as a reductase to produce GDP-6-deoxy-D-altro-heptose. Cjj1430 and Cjj1427 are the only members of the C3/C5 epimerases and C3/C5 epimerase/C4 reductase families shown to have activity on a heptose substrate and to exhibit only one of their two to three potential activities, respectively. Furthermore, we show that although the reductase WcaG is not part of the main pathway, its presence and its product affect the outcome of the pathway in a complex regulatory loop involving Cjj1427. This work provides the grounds for the elucidation of similar pathways found in other C. jejuni strains and other pathogens. It provides new molecular tools for the synthesis of carbohydrate antigens useful for vaccination and for the screening of enzymatic inhibitors that may have antibacterial effects.

Enhanced biofilm formation by Escherichia coli LPS mutants defective in Hep biosynthesis.

Lipopolysaccharide (LPS) is the major component of the surface of Gram-negative bacteria and its polysaccharide portion is situated at the outermost region. We investigated the relationship between the polysaccharide portion of LPS and biofilm formation using a series of Escherichia coli mutants defective in genes earlier shown to affect the LPS sugar compositions. Biofilm formation by a deep rough LPS mutant, the hldE strain, was strongly enhanced in comparison with the parental strain and other LPS mutants. The hldE strain also showed a phenotype of increased auto-aggregation and stronger cell surface hydrophobicity compared to the wild-type. Similar results were obtained with another deep rough LPS mutant, the waaC strain whose LPS showed same molecular mass as that of the hldE strain. Confocal laser scanning microscopy (CLSM) analysis and biofilm formation assay using DNase I revealed that biofilm formation by the hldE strain was dependent on extracellular DNA. Furthermore, a loss of flagella and an increase in amount of outer membrane vesicles in case of the hldE strain were also observed by transmission electron microscopy and atomic force microscopy, respectively. In addition, we demonstrated that a mutation in the hldE locus, which alters the LPS structure, caused changes in both expression and properties of several surface bacterial factors involved in biofilm formation and virulence. We suggest that the implication of these results should be considered in the context of biofilm formation on abiotic surfaces, which is frequently associated with nosocominal infections such as the catheter-associated infections.

Systematic synthesis of inhibitors of the two first enzymes of the bacterial heptose biosynthetic pathway: towards antivirulence molecules targeting lipopolysaccharide biosynthesis.

L-Heptoses (L-glycero-D-manno-heptopyranoses) are constituents of the inner core of lipolysaccharide (LPS), a molecule playing key roles in the mortality of many infectious diseases as well as in the virulence of many human pathogens. The inhibition of the first enzymes of the bacterial heptose biosynthetic pathway is an almost unexplored field to date although it appears to be a very novel way for the development of antivirulence drugs. We report the synthesis of a series of D-glycero-D-manno-heptopyranose 7-phosphate (H7P) analogues and their inhibition properties against the isomerase GmhA and the the kinase HldE, the two first enzymes of the bacterial heptose biosynthetic pathway. The heptose structures have been modified at the 1-, 2-, 6- and 7-positions to probe the importance of the key structural features of H7P that allow a tight binding to the target enzymes; H7P being the product of GmhA and the substrate of HldE, the second objective was to find structures that could simultaneously inhibit both enzymes. We found that GmhA and HldE were extremely sensitive to structural modifications at the 6- and 7- positions of the heptose scaffold. To our surprise, the epimeric analogue of H7P displaying a D-glucopyranose configuration was found to be the best inhibitor of both enzymes but also the only molecule of this series that could inhibit GmhA (IC(50)=34 µM) and HldE (IC(50)=9.4 µM) in the low micromolar range. Noteworthy, this study describes the first inhibitors of GmhA ever reported, and paves the way to the design of a second generation of molecules targeting the bacterial virulence.

Structural and kinetic characterization of the LPS biosynthetic enzyme D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) from Escherichia coli.

Lipopolysaccharide is a major component of the outer membrane of gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting gram-negative bacterial infection.

Structural studies of the O-antigens of Yersinia pseudotuberculosis O:2a and mutants thereof with impaired 6-deoxy-D-manno-heptose biosynthesis pathway.

The full structure of the long- and short-chain O-antigen of Yersinia pseudotuberculosis O:2a containing two uncommon deoxy sugars, abequose and 6-deoxy-d-manno-heptose (6dmanHep), was established, for the first time, by sugar analysis, NMR spectroscopy, and high-resolution ESIMS. Similar structural studies were also performed on two O:2a mutants with single disruption of 6dmanHep synthesis pathway genes each, which synthesize modified long-chain (dmhA mutant) and short-chain (both dmhA and dmhB mutants) O-antigens with 6dmanHep replaced by its putative biosynthetic precursor, D-glycero-D-manno-heptose.

The biosynthesis and biological role of 6-deoxyheptose in the lipopolysaccharide O-antigen of Yersinia pseudotuberculosis.

Yersinia pseudotuberculosis O:2a harbours 6-deoxy-d-manno-heptose in its O-antigen. The biological function of 6-deoxyheptose and its role in virulence is unknown and its biosynthetic pathway has not been demonstrated experimentally. Here, we show that dmhA and dmhB are necessary for 6-deoxyheptose biosynthesis in Y. pseudotuberculosis. Their disruption resulted in the lack of 6-deoxyheptose in the O-unit and its replacement by d-glycero-d-manno-heptose, thus indicating relaxed specificity of the glycosyltransferases, polymerase and ligase involved in lipopolysaccharide synthesis. The dmhB mutant exhibited a lower content in ketooctonic acid (Ko)-containing core molecules and reduced ligation and polymerization of the O-unit. We also show that Tyr128 is essential for activity of DmhB, and that DmhB functions as an oligomer, based on the dominant negative effect of overexpression of DmhB Y128F in dmhA. Moreover, we demonstrate that 6-deoxyheptose is important for virulence-related functions of the outer membrane and its appendages in vitro, such as barrier function against bile salts, polymyxin and novobiocin, and flagella-mediated motility. Although both mutants colonized the mouse ceacum as well as the wild type, the dmhB mutant was impaired for colonization of the liver, suggesting that DmhB represents a potential therapeutic target.