ADP heptose, a novel pathogen-associated molecular pattern identified in Helicobacter pylori.

The gastric pathogen Helicobacter pylori activates the NF-κB pathway in human epithelial cells via the recently discovered α-kinase 1 TRAF-interacting protein with forkhead-associated domain (TIFA) axis. We and others showed that this pathway can be triggered by heptose 1,7-bisphosphate (HBP), an LPS intermediate produced in gram-negative bacteria that represents a new pathogen-associated molecular pattern (PAMP). Here, we report that our attempts to identify HBP in lysates of H. pylori revealed surprisingly low amounts, failing to explain NF-κB activation. Instead, we identified ADP-glycero-ß-D-manno-heptose (ADP heptose), a derivative of HBP, as the predominant PAMP in lysates of H. pylori and other gram-negative bacteria. ADP heptose exhibits significantly higher activity than HBP, and cells specifically sensed the presence of the ß-form, even when the compound was added extracellularly. The data lead us to conclude that ADP heptose not only constitutes the key PAMP responsible for H. pylori-induced NF-κB activation in epithelial cells, but it acts as a general gram-negative bacterial PAMP.-Pfannkuch, L., Hurwitz, R., Traulsen, J., Sigulla, J., Poeschke, M., Matzner, L., Kosma, P., Schmid, M., Meyer, T. F. ADP heptose, a novel pathogen-associated molecular pattern identified in Helicobacter pylori.

Alpha-kinase 1 is a cytosolic innate immune receptor for bacterial ADP-heptose.

Immune recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors often activates proinflammatory NF-κB signalling1. Recent studies indicate that the bacterial metabolite D-glycero-ß-D-manno-heptose 1,7-bisphosphate (HBP) can activate NF-κB signalling in host cytosol2-4, but it is unclear whether HBP is a genuine PAMP and the cognate pattern recognition receptor has not been identified. Here we combined a transposon screen in Yersinia pseudotuberculosis with biochemical analyses and identified ADP-ß-D-manno-heptose (ADP-Hep), which mediates type III secretion system-dependent NF-κB activation and cytokine expression. ADP-Hep, but not other heptose metabolites, could enter host cytosol to activate NF-κB. A CRISPR-Cas9 screen showed that activation of NF-κB by ADP-Hep involves an ALPK1 (alpha-kinase 1)-TIFA (TRAF-interacting protein with forkhead-associated domain) axis. ADP-Hep directly binds the N-terminal domain of ALPK1, stimulating its kinase domain to phosphorylate and activate TIFA. The crystal structure of the N-terminal domain of ALPK1 and ADP-Hep in complex revealed the atomic mechanism of this ligand-receptor recognition process. HBP was transformed by host adenylyltransferases into ADP-heptose 7-P, which could activate ALPK1 to a lesser extent than ADP-Hep. ADP-Hep (but not HBP) alone or during bacterial infection induced Alpk1-dependent inflammation in mice. Our findings identify ALPK1 and ADP-Hep as a pattern recognition receptor and an effective immunomodulator, respectively.

Chemoenzymatic synthesis of ADP-d-glycero-ß-d-manno-heptose and study of the substrate specificity of HldE.

An efficient one-pot three enzymes strategy for chemoenzymatic synthesis of ADP-d-glycero-ß-d-manno-heptose (ADP-d, d-heptose) was reported using chemically synthesized d, d-heptose-7-phosphate and the ADP-d, d-heptose biosynthetic enzymes HldE and GmhB. Moreover, the result of investigating substrate specificity of the kinase action of HldE revealed that HldE had highly restricted substrate specificity towards structurally modified heptose-7-phosphate analogs.

The crystal structure of the Y140F mutant of ADP-L-glycero-D-manno-heptose 6-epimerase bound to ADP-beta-D-mannose suggests a one base mechanism.

Bacteria synthesize a wide array of unusual carbohydrate molecules, which they use in a variety of ways. The carbohydrate L-glycero-D-manno-heptose is an important component of lipopolysaccharide and is synthesized in a complex series of enzymatic steps. One step involves the epimerization at the C6'' position converting ADP-D-glycero-D-manno-heptose into ADP-L-glycero-D-manno-heptose. The enzyme responsible is a member of the short chain dehydrogenase superfamily, known as ADP-L-glycero-D-manno-heptose 6-epimerase (AGME). The structure of the enzyme was known but the arrangement of the catalytic site with respect to the substrate is unclear. We now report the structure of AGME bound to a substrate mimic, ADP-beta-D-mannose, which has the same stereochemical configuration as the substrate. The complex identifies the key residues and allows mechanistic insight into this novel enzyme.

Cytoplasmic Escherichia coli ADP sugar pyrophosphatase binds to cell membranes in response to extracellular signals as the cell population density increases.

ADP sugar pyrophosphatase (AspP) is a member of the 'Nudix' (Nucleoside diphosphate linked to some other moiety X) hydrolase family of enzymes that catalyzes the hydrolytic breakdown of ADP-glucose (ADPG) linked to glycogen biosynthesis. In a previous work, we showed that AspP activity is strongly enhanced by both glucose-1,6-bisphosphate and nucleotide-sugars, and by macromolecular crowding. In this work, we show that AspP binds to cell membranes as the bacterial population density increases, c. 30% of the total enzyme remaining membrane associated as glycogen depletes during the stationary phase. This process is not dependent on the stationary transcription factor RpoS, the producer of the bacterial quorum-sensing autoinducer 2 (LuxS), the presence of glycogen granules or glucose availability, but is stimulated by small soluble heat-labile molecule(s) occurring in cell-free spent supernatants of stationary cultures that are acid stabile and base labile. These data further point to AspP as a highly regulated enzyme, and provide a first set of evidences indicating that glycogen metabolism is subjected to regulation by intercellular communication in Escherichia coli.

Intermediate release by ADP-L-glycero-D-manno-heptose 6-epimerase.

ADP-l-glycero-d-manno-heptose 6-epimerase (HldD or AGME, formerly RfaD) catalyzes the interconversion of ADP-beta-d-glycero-d-manno-heptose (ADP-d,d-Hep) and ADP-beta-l-glycero-d-manno-heptose (ADP-l,d-Hep). The latter compound provides the heptose moiety that is used in lipopolysaccharide biosynthesis by Gram-negative bacteria. Several lines of evidence suggest that the enzyme uses a direct oxidation/reduction mechanism involving a tightly bound NADP+ cofactor. An initial oxidation at C-6'' gives a 6''-keto intermediate, and a subsequent reduction on the opposite face of the carbonyl group generates the epimeric product. The reorientation required for the nonstereoselective reduction could take place within a single active site, or it could involve the release of the intermediate and rebinding in an altered conformation. To distinguish between these possibilities, two isotopically labeled substrates (ADP-d,d-Hep) were prepared that contained 18O and 2H isotopes at C-7'' and C-6'', respectively. A crossover experiment was used to determine whether unlabeled or doubly labeled products were formed upon epimerization of a mixture of the two singly labeled compounds. After an initial epimeric equilibrium was reached, no crossover could be detected, indicating that intermediate release is not intrinsic to the overall mechanism. After extended incubation, however, scrambling of the labels could be detected, indicating that a low background rate of intermediate release does occur. To directly detect the release of the intermediate, the labeled compounds were independently epimerized in the presence of a ketone-trapping reagent, phenylhydrazine. The corresponding phenylhydrazones were identified by mass spectrometry, and the absence of any 2H isotope in the adduct obtained from the deuterated starting compound confirmed that the oxidation had occurred at C-6''.

Escherichia coli AspP activity is enhanced by macromolecular crowding and by both glucose-1,6-bisphosphate and nucleotide-sugars.

Escherichia coli ADP-sugar pyrophosphatase (AspP) is a "Nudix" hydrolase that catalyzes the hydrolytic breakdown of ADP-glucose linked to glycogen biosynthesis. Moderate increases of AspP activity in the cell are accompanied by significant reductions of the glycogen content. In vitro analyses showed that AspP activity is strongly enhanced by macromolecular crowding and by both glucose-1,6-bisphosphate and nucleotide-sugars, providing a first set of indicative evidences that AspP is a highly regulated enzyme. To our knowledge, AspP is the sole bacterial enzyme described to date which is activated by both G1,6P(2) and nucleotide-sugars.

A two-base mechanism for Escherichia coli ADP-L-glycero-D-manno-heptose 6-epimerase.

ADP-l-glycero-d-manno-heptose 6-epimerase (HldD or AGME, formerly RfaD) catalyzes the inversion of configuration at C-6' ' of the heptose moiety of ADP-d-glycero-d-manno-heptose and ADP-l-glycero-d-manno-heptose. The epimerase HldD operates in the biosynthetic pathway of l-glycero-d-manno-heptose, which is a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. Previous studies support a mechanism in which HldD uses its tightly bound NADP+ cofactor to oxidize directly at C-6' ', generating a ketone intermediate. A reduction of the ketone from the opposite face then occurs, generating the epimeric product. How the epimerase is able access both faces of the ketone intermediate with correct alignment of the three required components, NADPH, the ketone carbonyl, and a catalytic acid/base residue, is addressed here. It is proposed that the epimerase active site contains two catalytic pockets, each of which bears a catalytic acid/base residue that facilitates reduction of the C-6' ' ketone but leads to a distinct epimeric product. The ketone carbonyl may access either pocket via rotation about the C-5' '-C-6' ' bond of the sugar nucleotide and in doing so presents opposing faces to the bound cofactor. Evidence in support of the two-base mechanism is found in studies of two single mutants of the Escherichia coli K-12 epimerase, Y140F and K178M, both of which have severely compromised epimerase activities that are more than 3 orders of magnitude lower than that of the wild type. The catalytic competency of these two mutants in promoting redox chemistry is demonstrated with an alternate catalytic activity that requires only one catalytic base: dismutation of a C-6' ' aldehyde substrate analogue (ADP-beta-d-manno-hexodialdose) to an acid and an alcohol (ADP-beta-d-mannuronic acid and ADP-beta-d-mannose). This study identifies the two catalytic bases as tyrosine 140 and lysine 178. A one-step enzymatic conversion of mannose into ADP-beta-mannose is also described and used to make C-6' '-substituted derivatives of this sugar nucleotide.

An in vitro screen of bacterial lipopolysaccharide biosynthetic enzymes identifies an inhibitor of ADP-heptose biosynthesis.

The lipopolysaccharide (LPS)-rich outer membrane of gram-negative bacteria provides a protective barrier that insulates these organisms from the action of numerous antibiotics. Breach of the LPS layer can therefore provide access to the cell interior to otherwise impermeant toxic molecules and can expose vulnerable binding sites for immune system components such as complement. Inhibition of LPS biosynthesis, leading to a truncated LPS molecule, is an alternative strategy for antibacterial drug development in which this vital cellular structure is weakened. A significant challenge for in vitro screens of small molecules for inhibition of LPS biosynthesis is the difficulty in accessing the complex carbohydrate substrates. We have optimized an assay of the enzymes required for LPS heptose biosynthesis that simultaneously surveys five enzyme activities by using commercially available substrates and report its use in a small-molecule screen that identifies an inhibitor of heptose synthesis.

Efficient chemoenzymatic synthesis of ADP-D-glycero-beta-D-manno-heptose and a mechanistic study of ADP-L-glycero-D-manno-heptose 6-epimerase.

[reaction: see text] A chemoenzymatic synthesis of ADP-D-glycero-beta-D-manno-heptose (ADP-D,D-Hep) is described in which D,D-Hep 7-phosphate is converted to ADP-D,D-Hep by two biosynthetic enzymes. This strategy allows access to the 6''-deuterated analogue, which upon incubation with the epimerase showed complete retention of the isotopic label at the 6''-position. This provides evidence for a direct oxidation mechanism in which the hydride initially transferred to the NADP+ cofactor is subsequently returned to the same carbon in a nonstereospecific manner.

Synthesis of unnatural sugar nucleotides and their evaluation as donor substrates in glycosyltransferase-catalyzed reactions.

New unnatural sugar nucleotides, UDP-Fuc and CDP-Fuc were synthesized from fucose-beta-1-phosphate and nucleotide monophosphates activated as morpholidates. Furthermore, a nucleotide analogue was prepared by phosphorylation of 1-(beta-D-ribofuranosyl)cyanuric acid, itself obtained as a protected derivative by condensation of the persilylated derivative of cyanuric acid with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose in 74% yield. This phosphate activated according to the same procedure was condensed with fucose-beta-1-phosphate, affording a new sugar nucleotide conjugate (NDP-Fuc) which was evaluated together with UDP-Fuc, CDP-Fuc and ADP-Fuc, as fucose donors in alpha-(1-->4/3)-fucosyltransferase (FucT-III) catalyzed reaction. Fucose transfer could be observed with each of the donors and kinetic parameters were determined using a fluorescent acceptor substrate. Efficiency of the four analogues towards FucT-III was in the following order: UDP-Fuc=ADP-Fuc>NDP-Fuc>CDP-Fuc. According to the same strategy ADP-GlcNAc was prepared from AMP-morpholidate and N-acetylglucosamine-alpha-1-phosphate; tested as a glucosaminyl donor towards Neisseria meningitidis N-acetylglucosaminyl transferase (LgtA), ADP-GlcNAc was recognized with 0.1% efficiency as compared with UDP-GlcNAc, the natural donor substrate.

Biosynthesis pathway of ADP-L-glycero-beta-D-manno-heptose in Escherichia coli.

The steps involved in the biosynthesis of the ADP-L-glycero-beta-D-manno-heptose (ADP-L-beta-D-heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated. In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP-D-beta-D-heptose and have biochemically characterized the reaction products by high-performance anion-exchange chromatography. We have also constructed a deletion in a novel gene, gmhB (formerly yaeD), which results in the formation of an altered LPS core. This mutation confirms that the GmhB protein is required for the formation of ADP-D-beta-D-heptose. Our results demonstrate that the synthesis of ADP-D-beta-D-heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional D-beta-D-heptose 7-phosphate kinase/D-beta-D-heptose 1-phosphate adenylyltransferase), and GmhB (D,D-heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate. A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP-L-beta-D-heptose precursor utilized in the assembly of inner core LPS.

Characterization of the physiological substrate for lipopolysaccharide heptosyltransferases I and II.

L-Glycero-D-manno-heptopyranose is a characteristic compound of many lipopolysaccharide (LPS) core structures of Gram-negative bacteria. In Escherichia coli two heptosyltransferases, namely WaaC and WaaF, are known to transfer L-glycero-D-manno-heptopyranose to Re-LPS and Rd(2)-LPS, respectively. It had been proposed that both reactions involve ADPL-glycero-D-manno-heptose as a sugar donor; however, the structure of this nucleotide sugar had never been completely elucidated. In the present study, ADPL-glycero-D-manno-heptose was isolated from a heptosyltransferase-deficient E. coli mutant, and its structure was determined by nuclear magnetic resonance spectroscopy and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry as ADPL-glycero-beta-D-manno-heptopyranose. This compound represented the sole constituent of the bacterial extract that was accepted as a sugar donor by heptosyltransferases I and II in vitro.

Cloning and characterization of a new member of the Nudix hydrolases from human and mouse.

Proteins containing the Nudix box "GX(5)EX(7)REUXEEXGU" (where U is usually Leu, Val, or Ile) are Nudix hydrolases, which catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Here we report cloning and characterization of a human cDNA encoding a novel nudix hydrolase NUDT5 for the hydrolysis of ADP-sugars. The deduced amino acid sequence of NUDT5 contains 219 amino acids, including a conserved Nudix box sequence. The recombinant NUDT5 was expressed in Escherichia coli and purified to near homogeneity. At the optimal pH of 7, the purified recombinant NUDT5 catalyzed hydrolysis of two major substrates ADP-ribose and ADP-mannose with K(m) values of 32 and 83 microM, respectively; the V(max) for ADP-mannose was about 1.5 times that with ADP-ribose. The murine NUDT5 homolog was also cloned and characterized. mNudT5 has 81% amino acid identity to NUDT5 with catalytic activities similar to NUDT5 under the optimal pH of 9. Both NUDT5 and mNudT5 transcripts were ubiquitously expressed in tissues analyzed with preferential abundance in liver. The genomic structures of both NUDT5 and mNudT5 were determined and located on human chromosome 10 and mouse chromosome 2, respectively. The role of NUDT5 in maintaining levels of free ADP-ribose in cells is discussed.

Cloning, expression and characterization of YSA1H, a human adenosine 5'-diphosphosugar pyrophosphatase possessing a MutT motif.

The human homologue of the Saccharomyces cerevisiae YSA1 protein, YSA1H, has been expressed as a thioredoxin fusion protein in Escherichia coli. It is an ADP-sugar pyrophosphatase with similar activities towards ADP-ribose and ADP-mannose. Its activities with ADP-glucose and diadenosine diphosphate were 56% and 20% of that with ADP-ribose respectively, whereas its activity towards other nucleoside 5'-diphosphosugars was typically 2-10%. cADP-ribose was not a substrate. The products of ADP-ribose hydrolysis were AMP and ribose 5-phosphate. K(m) and k(cat) values with ADP-ribose were 60 microM and 5.5 s(-1) respectively. The optimal activity was at alkaline pH (7.4-9.0) with 2.5-5 mM Mg(2+) or 100-250 microM Mn(2+) ions; fluoride was inhibitory, with an IC(50) of 20 microM. The YSA1H gene, which maps to 10p13-p14, is widely expressed in all human tissues examined, giving a 1.4 kb transcript. The 41.6 kDa fusion protein behaved as an 85 kDa dimer on gel filtration. After cleavage with enterokinase, the 24.4 kDa native protein fragment ran on SDS/PAGE with an apparent molecular mass of 33 kDa. Immunoblot analysis with a polyclonal antibody raised against the recombinant YSA1H revealed the presence of a protein of apparent molecular mass 33 kDa in various human cells, including erythrocytes. The sequence of YSA1H contains a MutT sequence signature motif. A major proposed function of the MutT motif proteins is to eliminate toxic nucleotide metabolites from the cell. Hence the function of YSA1H might be to remove free ADP-ribose arising from NAD(+) and protein-bound poly- and mono-(ADP-ribose) turnover to prevent the occurrence of non-enzymic protein glycation.

Adenosine diphosphate ribulose in human erythrocytes: a new metabolite with membrane binding properties.

Incubation of ADPribose with yeast phosphoriboisomerase resulted in the formation of an adenylic nucleotide that was identified with ADPribulose by mass spectrometry. Synthesis of [32P]ADPribulose from [32P]NAD+ by the combined activities of commercial NAD+ glycohydrolase and phosphoriboisomerase allowed us to use it as a labeled internal standard throughout the procedure of purification from trichloroacetic acid extracts of human red blood cells. ADPribulose was purified by means of three sequential reverse phase HPLC separations and its concentration in human erythrocytes was estimated to be 0.11 +/- 0.1 microM. Unsealed erythrocyte ghosts did not transform ADPribulose, which bound to specific membrane proteins with a trichloroacetic and formic acid-resistant binding. The labeled proteins were identified as spectrin, bands 3, 4.1, 4.2 and Glyceraldehyde 3-phosphate dehydrogenase on the basis of their relative mobilities on SDS-PAGE.

Isolation of adenosine 5'-diphosphate-L-glycero-D-mannoheptose, the assumed substrate of heptose transferase(s), from Salmonella minnesota R595 and Shigella sonnei Re mutants.

From heptose transferase-less Re mutants of Salmonella minnesota and Shigella sonnei, a mixture of nucleotide-linked heptoses was isolated. After paper chromatography in different solvent systems, ADP derivatives of D-glycero-D-mannoheptose and L-glycero-D-mannoheptose could be isolated in pure form. The structure of ADP-L-glycero-D-mannoheptose was verified by analytical methods and by transformation of ADP-D-glycero-D-mannoheptose with ADP-D-glycero-D-mannoheptose-6-epimerase.

Isolation of adenosine 5'-diphosphate-D-glycero-D-mannoheptose. An intermediate in lipopolysaccharide biosynthesis of Shigella sonnei.

From a Shigella sonnei R mutant which incorporates into its cell wall lipopolysaccharide D-glycero-D-mannoheptose and contains no L-glycero-D-mannoheptose, a nucleotide-linked sugar was isolated and identified as adenosine 5'-diphosphate-D-glycero-d-mannoheptose by chemical and chromatographic analysis. This intermediary compound is assumed to play a role in heptose biosynthesis of Enterobacteria.

The enthalpy of protolysis of liver alcohol dehydrogenase upon binding nicotinamide adenine dinucleotide.

The binding of NAD+, NADH, and ADP-ribose to horse liver alcohol dehydrogenase has been studied calorimetrically as a function of pH at 25 degrees C. The enthalpy of NADH binding is 0 +/- 0.5 kcal mol-1 in the pH range 6 to 8.6. The enthalpy of NAD+ binding, however, varies with pH in a sigmoidal fashion and is -4.0 kcal mol(NAD)-1 at pH 6.0 and +4.5 kcal mol(NAD)-1 at pH 8.6 with an apparent pKa of 7.6 +/- 0.2. The enthalpy of proton ionization of the group on the enzyme is calculated to be in the range 8.8 to 9.8 kcal mol(H+)-1. In conjunction with the available thermodynamic data on the ionization of zinc-bound water in model compounds, it is concluded that the group with a pKa of 9.8 in the free enzyme and 7.6 in the enzyme . NAD+ binary complex is, most likely, the zinc-bound water molecule. Our studies with zinc-free enzyme provide further evidence for this conclusion. Therefore, the processes involving a conformational change of the enzyme upon NAD+ binding and the suggested mechanism of subsequent quenching of the fluorescence of Trp-314 implicating the participation of an ionized tyrosine group must be re-evaluated in the light of this thermodynamic study.