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.

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.

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.

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.

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.

Spermine-induced variations in the adenosine 5'-diphosphate ribosylation patterns of nuclear proteins from rat liver and hepatoma.

Rat liver and hepatoma nuclei were incubated in vitro with [3H]nicotinamide adenine dinucleotide to allow synthesis of a polymer of adenosine diphosphoribose subunits joined in an 1',2' ribose-ribose linkage. The addition of 1 mM spermine altered the adenosine 5'-diphosphate (ADP) ribosylation patterns of nuclear proteins in hepatoma, host liver, and regenerating liver. Spermine-treated nuclei showed a greater incorporation of ADP-ribose into H1 histones and nonhistone nuclear proteins with isoelectric points between pH 3.0 and 6.0 when separated on polyacrylamide gels. Conversely, a large reduction in ADP ribosylation was seen in core histones (H2A, H2B, and H3) from the same nuclei. The proportion of ADP-ribose incorporated into histones was reduced in the nuclei from proliferating cells relative to their respective control livers. These results imply that polyamines, which are higher in concentration in rapidly dividing cells, may elicit a regulatory function by causing the preferential ADP ribosylation of H1 histones, as well as the more acidic of the nuclear proteins.

The mechanism of ADP-ribosylation of elongation factor 2 catalyzed by fragment A from diphtheria toxin.

Measurements of the initial rate of ADP-ribosylation of elongation factor 2 (EF-2) catalyzed by Fragment A from diphtheria toxin support a sequential mechanism and suggest that the reaction proceeds through a central ternary complex involving Fragment A and the substrates, EF-2 and NAD. The Michaelis constants for EF-2 and NAD are 0.15 and 1.4 muM, respectively. As determined by equilibrium gel permeation, EF-2 does not bind Fragment A significantly, alone or in the presence of adenine, ADPribose, nicotinamide or NADH. Based on these and earlier results, we propose an ordered sequential mechanism for the reaction; the sequence of binding of substrates is NAD, followed by EF-2.

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.

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.

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.

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''.

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.

Immobilization of reticulocyte elongation factor EF-2.

Conditions are described whereby the ADP-ribosylation (from NAD+) of reticulocyte elongation factor EF-2, catalyzed by diphtheria toxin, is essentially complete and whereby the reverse of this process may be carried out with recovery of 60--70% of the original EF-2 activity. Both reactions proceed well at room temperature. The reverse reaction is much slower than the ADP-ribosylation process and requires high nicotinamide concentrations. For the reverse reaction to occur at a significant rate it is necessary to lower the pH to 6.5 (from the 7.5 used for the forward reaction). NAD+ covalently linked to agarose may replace NAD+ in the diphtheria toxin reaction. The characteristics of this reaction are similar to those of the reaction employing free NAD+ except that the velocity is reduced and the concentration of NAD+ moieties greatly increased. NAD+ immobilized on agarose through the C-8 of the adenine ring is a superior substrate compared with NAD+ linked to agarose via its periodate-oxidized ribose moieties. Preliminary experiments indicate that reversal of this latter reaction with recovery of biological activity may be possible.

[Protein-bound mono (adenosine-diphosphate-ribose) levels during the cell cycle of the slime mold Physarum polycephalum]. / Protein-gebundene Mono(Adenosindiphosphat-Ribose)-Spiegel während des Zellzyklus von Physarum polycephalum.

In Physarum polycephalum two fractions of ADPR-protein conjugates could be differentiated on the basis of their susceptibility towards hydroxylamine. Quantitation during the cell cycle revealed independent synthesis of the two species, the NH2OH-resistant fraction being formed during S phase, while the NH2OH-sensitive conjugate increased sharply at the S/G2 boundary. These findings indicated that nuclear ADP-ribosylation reactions are more than one function.