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1.
In previous studies we described enzyme(s) from Escherichia coli that transfer two 3-deoxy-D-manno-octulosonate (KDO) residues from two CMP-KDO molecules to a tetraacyldisaccharide-1,4'-bis-phosphate precursor of lipid A, termed lipid IVA (Brozek, K. A., Hosaka, K., Robertson, A. D., and Raetz, C. R. H. (1989) J. Biol. Chem. 264, 6956-6966). The product, designated (KDO)2-IVA, can be prepared in milligram quantities and/or radiolabeled with 32P at position 4' of the IVA moiety. We now demonstrate the presence of enzymes in E. coli extracts that transfer laurate and/or myristate residues from lauroyl or myristoyl-acyl carrier protein (ACP) to (KDO)2-IVA. Thioesters of coenzyme A are not substrates. The cytosolic fraction catalyzes rapid acylation with lauroyl-ACP, but not with myristoyl, R-3-hydroxymyristoyl, palmitoyl, or palmitoleoyl-ACP. The membrane fraction transfers both laurate and myristate to (KDO)2-IVA. Evidence for the enzymatic acylation of (KDO)2-IVA is provided by (a) conversion of [4'-32P](KDO)2-IVA to more rapidly migrating products in the presence of the appropriate acyl-ACP, (b) incorporation of [1-14C]laurate or [1-14C]myristate into these metabolites in the presence of (KDO)2-IVA, (c) fast atom bombardment-mass spectrometry, and (d) 1H NMR spectroscopy. At protein concentrations less than 0.5 mg/ml, the acylation of (KDO)2-IVA by the cytoplasmic fraction is absolutely dependent upon the addition of exogenous acyl-ACP. These acyltransferases cannot utilize lipid IVA as a substrate, demonstrating that they possess novel KDO recognition domains. The unusual substrate specificity of these enzymes provides compelling evidence for their involvement in lipid A biosynthesis. Depending on the conditions it is possible to acylate (KDO)2-IVA with 1 or 2 lauroyl residues, with 1 or 2 myristoyl residues, or with 1 of each.  相似文献   

2.
The enzyme 3-deoxy-D-manno-octulosonic acid (Kdo) transferase is encoded by the kdtA gene of Escherichia coli and plays a key role in lipopolysaccharide biosynthesis. It transfers Kdo from CMP-Kdo to lipid A or its tetraacyldisaccharide-1,4'-bisphosphate precursor, lipid IVA. Using a strain that overproduces the transferase approximately 500-fold, we have purified the enzyme to near homogeneity. The subunit molecular mass is approximately 43 kDa. Activity is stimulated by Triton X-100, is maximal at pH 7, but does not require Mg2+. The apparent Km values for lipid IVA and CMP-Kdo are 52 and 88 microM, respectively. Vmax is 15-18 mumol/min/mg when both substrates are added near saturation at pH 8. The purified enzyme transfers 2 Kdo residues to lipid A precursors or analogs bearing four to six fatty acyl chains and a 4'-monosphosphate moiety. Activity is inhibited by polymixin B and Re endotoxin. At low Kdo concentrations small amounts of the intermediate, (Kdo)1-IVA, accumulate. When this substance is isolated and incubated with purified enzyme in the presence of CMP-Kdo, it is converted to (Kdo)2-IVA. Formation of (Kdo)1-IVA is also observed when purified enzyme is incubated with (Kdo)2-IVA and 5 mM CMP, demonstrating that Kdo transfer is reversible. In summary, Kdo transferase consists of a single bifunctional polypeptide that incorporates the 2 innermost Kdo residues common to all lipopolysaccharide molecules in E. coli.  相似文献   

3.
4.
5.
An autoradiographic assay applicable to colonies immobilized on filter paper was developed for obtaining temperature-sensitive mutants of Escherichia coli defective in the transfer of 3-deoxy-D-manno-octulosonic acid (KDO) from CMP-KDO to a tetraacyldisaccharide 1,4'-bisphosphate precursor of lipid A, designated lipid IVA. Cell-free extracts from two mutants found in a population of 30,000 mutagen-treated cells showed normal KDO transferase activity when assayed at 30 degrees C, but almost no activity at 42 degrees C. The mutation was mapped by mating one of the mutants with different Hfr strains and analyzing genetic linkage of KDO transferase activity to selectable markers. The lesion was located to a position between 80 and 84 min on the E. coli chromosome. A plasmid from the Clarke and Carbon collection (Clarke, L., and Carbon, J. (1976) Cell 9, 91-99), pLC17-24, known to contain genes from the rfa region (81 min), was shown to overexpress KDO transferase activity 4-5 times and to correct the mutation when the plasmid was conjugated into the mutant strains. The KDO transferase gene, designated kdtA, was subcloned from pLC17-24 into a multicopy vector. The resulting plasmid, pCL3, overproduced transferase activity approximately 100-fold. The kdtA gene was shown to code for a 43-kDa polypeptide, as judged by radiolabeling of minicells. Its DNA sequence was determined. The results demonstrate that overexpression of this single gene product greatly stimulates the incorporation of two stereochemically distinct KDO residues during lipopolysaccharide biosynthesis in extracts of E. coli.  相似文献   

6.
Antibacterial agents which specifically inhibit CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase activity were used to block the incorporation of 3-deoxy-D-manno-octulosonate (KDO) into lipopolysaccharide. Lipopolysaccharide synthesis ceased, molecules similar in structure to lipid A accumulated, and bacterial growth ceased following addition of such agents to cultures of Salmonella typhimurium and Escherichia coli. Although four major species of lipid A accumulated in S. typhimurium, their kinetics of accumulation were different. The least polar of the major species was IVA [O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1----6)-2-amino-2-deoxy-a lph a- D-glucose, acylated at positions 2, 3, 2', and 3' with beta-hydroxymyristoyl groups and bearing phosphates at positions 1 and 4'], a molecule previously isolated from bacteria containing a kdsA mutation (C. R. H. Raetz, S. Purcell, M. V. Meyer, N. Qureshi, and K. Takayama, J. Biol. Chem. 260:16080-16088, 1985). Species IVA accumulated first and to the greatest extent following addition of the inhibitor, with other more polar derivatives appearing only after IVA attained half its maximal level. In contrast, only two major species of precursor accumulated in E. coli following addition of the inhibitor. One of these species was identical to IVA from S. typhimurium on the basis of chemical composition, fast atom bombardment mass spectroscopy, and comigration on Silica Gel H, and it also accumulated prior to a more polar species of related structure. We conclude that the addition of KDO to precursor species IVA is the major pathway of lipid A-KDO formation in both S. typhimurium LT2 and E. coli and that accumulation of the more polar species lacking KDO only occurs in response to accumulation of species IVA following inhibition of the normal pathway.  相似文献   

7.
The cell envelope fraction of Salmonella typhimurium contains an enzyme system which catalyzes transfer of 3-deoxyoctulosonate (KDO) from CMP-KDO to an incomplete, KDO-deficient precursor of lipid A. The enzyme system is firmly membrane-bound, but has been solubilized by treatment with nonionic detergent at alkaline pH and partially purified. Both the particulate and partially purified fractions catalyzed formation of a single reaction product containing 2 residues of KDO. Periodate oxidation of the purified product permitted tentative identification of the KDO disaccharide structure as KDO2-4KDO.  相似文献   

8.
Analogs of 3-deoxy-D-manno-octulosonate (KDO) were designed to inhibit CTP:CMP-KDO cytidylyltransferase (CMP-KDO synthetase). Since these analogs lacked whole-cell antibacterial activity, a permeabilized-cell method was developed to measure intracellular compound activity directly. The method employed a mutant of Salmonella typhimurium defective in KDO-8-phosphate synthetase (kdsA), which accumulated lipid A precursor at 42 degrees C. Cells permeabilized with 1% toluene were used to evaluate inhibitor effect on [3H]KDO incorporation into preformed lipid A precursor. KDO incorporation proceeded through the enzymes CMP-KDO synthetase and CMP-KDO:lipid A KDO transferase. Optimum KDO incorporation occurred between pH 8 and 9 and required CTP, prior lipid A precursor accumulation, and a functional kdsB gene product, CMP-KDO synthetase. The apparent Km for KDO in this coupled system at pH 7.6 was 1.38 mM. The reaction products isolated and characterized contained 1 and 2 KDO residues per lipid A precursor molecule. Several KDO analogs produced concentration-related reductions of KDO incorporation in toluenized cells with 50% inhibitory concentrations comparable to those obtained in purified CMP-KDO synthetase systems. Two compounds, 8-amino-2-deoxy-KDO (A-60478) and 8-aminomethyl-2-deoxy-KDO (A-60821), competitively inhibited KDO incorporation, displaying Kis of 4.2 microM for A-60478 and 2.5 microM for A-60821. These data indicated that the inactivity of the KDO analogs on intact bacteria was the result of poor permeation into cells rather than intracellular inactivation.  相似文献   

9.
Lipid A from the nitrogen-fixing bacterium Rhizobium leguminosarum displays many structural differences compared with lipid A of Escherichia coli. R. leguminosarum lipid A lacks the usual 1- and 4'-phosphate groups but is derivatized with a galacturonic acid substituent at position 4'. R. leguminosarum lipid A often contains an aminogluconic acid moiety in place of the proximal glucosamine 1-phosphate unit. Striking differences also exist in the secondary acyl chains attached to E. coli versus R. leguminosarum lipid A, specifically the presence of 27-hydroxyoctacosanoate and the absence of laurate and myristate in R. leguminosarum. Recently, we have found that lipid A isolated by pH 4.5 hydrolysis of R. leguminosarum cells is more heterogeneous than previously reported (Que, N. L. S., Basu, S. S., White, K. A., and Raetz, C. R. H. (1998) FASEB J. 12, A1284 (abstr.)). Lipid A species lacking the 3-O-linked beta-hydroxymyristoyl residue on the proximal unit contribute to this heterogeneity. We now describe a membrane-bound deacylase from R. leguminosarum that removes a single ester-linked beta-hydroxymyristoyl moiety from some lipid A precursors, including lipid X, lipid IVA, and (3-deoxy-D-manno-octulosonic acid)2-lipid IVA. The enzyme does not cleave E. coli lipid A or lipid A precursors containing an acyloxyacyl moiety on the distal glucosamine unit. The enzyme is not present in extracts of E. coli or Rhizobium meliloti, but it is readily demonstrable in membranes of Pseudomonas aeruginosa, which also contains a significant proportion of 3-O-deacylated lipid A species. Optimal reaction rates are seen between pH 5.5 and 6.5. The enzyme requires a nonionic detergent and divalent metal ions for activity. It cleaves the monosaccharide lipid X at about 5% the rate of lipid IVA and (3-deoxy-D-manno-octulosonic acid)2-lipid IVA. 1H NMR spectroscopy of the deacylase reaction product, generated with lipid IVA as the substrate, confirms unequivocally that the enzyme cleaves only the ester-linked beta-hydroxymyristoyl residue at the 3-position of the glucosamine disaccharide.  相似文献   

10.
Lipid A binding sites in membranes of macrophage tumor cells   总被引:14,自引:0,他引:14  
Lipopolysaccharide affects a variety of eukaryotic cells and mammalian organisms. These actions are involved in the pathogenesis of Gram-negative septicemia. Many of the actions of lipopolysaccharide are believed to be caused by its active moiety, lipid A. Our laboratory has previously identified a bioactive lipid A precursor, termed lipid IVA (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16888), which can be labeled with 32P of high specific activity and purified. In this work we have used the labeled probe, 4'-32P-lipid IVA, to develop a novel assay for the specific binding of lipid IVA to whole cells. We have also demonstrated its use in a ligand blotting assay of immobilized cellular proteins. Using the whole cell assay, we show that 4'-32P-lipid IVA specifically binds to RAW 264.7 macrophage-like cultured cells. The binding is saturable, is inhibited with excess unlabeled lipid IVA, and is proteinase K-sensitive. It displays cellular and pharmacological specificity. Using the ligand blotting assay, we show that several RAW 264.7 cell proteins can bind 4'-32P-lipid IVA. The two principal binding proteins have Mr values of 31 and 95 kDa, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fractionation studies indicate that the 31-kDa protein is enriched in the nuclear fraction and may be a histone, whereas the 95-kDa protein is enriched in the membrane fraction. The binding assays that we have developed should lead to a clearer understanding of lipid A/animal cell interactions.  相似文献   

11.
Macrophage catabolism of lipid A is regulated by endotoxin stimulation   总被引:1,自引:0,他引:1  
Lipopolysaccharide (LPS) is a Gram-negative bacterial glycolipid that is believed to cause, by virtue of its stimulatory actions on macrophages and other eukaryotic cells, the life-threatening symptoms associated with Gram-negative infections. Macrophages both respond to and catabolically deactivate LPS. The lipid A moiety of LPS is responsible for the stimulatory actions of LPS on macrophages. We have previously developed methods employing a radiolabeled bioactive lipid A precursor, 4'-32P-lipid IVA, to study the interaction of this class of lipids with animal cells (Hampton, R. Y., Golenbock, D. T., and Raetz, C. R. H. (1988). J. Biol. Chem. 263, 14802-14807). In the current work, we have examined the uptake and catabolism of 4'-32P-lipid IVA by the RAW 264.7 cell line in serum-containing medium at physiological temperatures and have studied the effect of LPS stimulation on the ability of these cells to catabolize lipid IVA. RAW 264.7 macrophage-like cells avidly take up 4'-32P-lipid IVA under cell culture conditions at nanomolar concentrations. Uptake of lipid IVA was accompanied by lysosomal dephosphorylation of a fraction of the lipid to yield 4'-monophosphoryl lipid IVA. Chemically generated 4'-monophosphoryl lipid IVA was found to be substantially less bioactive than lipid IVA in the RAW cell, indicating that this catabolic dephosphorylation results in detoxification. In uptake experiments of 3-4 h duration, all metabolism of lipid IVA is blocked by ligands of the macrophage scavenger receptor. In longer experiments (24 h), both scavenger receptor-dependent and -independent uptake are responsible for the lysosomal catabolism of lipid IVA. Preincubation of RAW 264.7 cells with LPS caused dose-dependent inhibition of lipid IVA dephosphorylation. Sufficient LPS stimulation resulted in essentially complete inhibition of lipid IVA catabolism in both short- and long-term uptake experiments. This effect occurred at physiologically relevant concentrations of LPS (IC50 less than 1 ng/ml), and our data indicate that LPS-induced blockade of lipid IVA catabolism was due to the resultant physiological stimulation of the cells, and not inhibition of dephosphorylation by competition for uptake or enzymatic sites or by simple sequestration of labeled lipid IVA by LPS aggregates. We suggest that in the macrophage, LPS can modulate its own catabolism by virtue of its pharmacological properties. This effect of LPS could play a role in LPS pathophysiology as well as in macrophage biology.  相似文献   

12.
Arachidonic acid (AA) and its metabolites mediate many physiological processes including reproduction and endocrinology. The current study investigated effects of several inhibitors of AA cascade on steroidogenesis by rat corpus luteum cells in vitro. Dispersed luteal cells prepared from rat corpus luteum on day 6 of pseudopregnancy secreted progesterone (P4) in time-dependent and human chorinonic gonadotropin (hCG)-dependent fashion. Arachidonyl trifluoromethyl ketone, a preferential inhibitor of the type IVA phospholipase A(2) (PLA(2)-IVA), stimulated basal P4 secretion and had no influence on hCG-stimulated steroidogenesis. A novel and more specific inhibitor pyrrophenone inhibited hCG-induced P4 secretion. A cyclooxygenase inhibitor indomethacin did not affect basal secretion but inhibited hCG-stimulated secretion. Nordihydroguaiaretic acid tended to decrease basal P4 secretion and completely inhibited hCG-stimulated secretion. Obtained results suggest that AA metabolic cascade, which is triggered at least in part by PLA(2)-IVA activity, is potentially implicated in hCG-stimulated P4 secretory response in the rat corpus luteum.  相似文献   

13.
Spontaneous mutants of Salmonella typhimurium LT2 were selected for the ability to accumulate exogenous 3-deoxy-D-manno-octulosonate (KDO). Bacteria containing a gene (kdsA) which codes for a temperature-sensitive KDO-8-phosphate synthetase were plated at the restrictive temperature of 42 degrees C on medium containing 5 mM KDO. Since bacteria containing the kdsA lesion are unable to grow at 42 degrees C due to inhibition of lipopolysaccharide (LPS) synthesis and accumulation of lipid A precursor, this method allowed direct, positive selection of mutants capable of utilizing exogenous KDO for LPS synthesis. Spontaneous mutants, selected at a frequency of about 10(-6), required exogenous KDO for growth at 42 degrees C. The growth rate at 42 degrees C was nearly normal in the presence of 20 mM KDO and was directly proportional to KDO concentrations below 20 mM. Exogenous KDO also suppressed accumulation of lipid A precursor. The apparent Km for KDO accumulation was 23 mM, and the maximum rate of transport was calculated to be 505 pmol of KDO per min per 10(8) cells. Bacteria incorporated exogenous [3H]KDO exclusively into LPS, with less than 10% dilution in specific activity due to residual endogenous KDO synthesis. The mutation giving rise to the ability to accumulate exogenous KDO was extremely useful in the direct screening for new mutations in the kdsA gene after localized mutagenesis. Five mutations in kdsA were isolated, four of which were new alleles as determined by on fine-structure analysis. The ability to introduce labeled (3H, 13C, and 14C) KDO in vivo should simplify and extend the analysis of this critical metabolic pathway in gram-negative bacteria.  相似文献   

14.
The first preliminary structure of a surface lipooligosaccharide from Haemophilus ducreyi has been determined. The major oligosaccharide was released by mild acid hydrolysis and analyzed by liquid secondary ion and tandem mass spectrometry. The mass spectral data combined with composition and methylation analysis yielded the most probable structure; Gal1----4GlcNAc1----3Gal1----4Hep1----6Glc1----( Hep1----2Hep1----)3,4Hep1---- KDO, where the reducing terminal 3-deoxy-D-manno-octulosonic acid (or KDO) exists in an anhydro form. This anhydro species results from the elimination of a phosphate from C-4 of KDO during mild acid hydrolysis. The core heptose trisaccharide consists of L-glycero-D-manno-heptose, but analysis of the peracetylated sugars indicated that the 1,4-linked heptose is likely D-glycero-D-manno-heptose. The monoclonal antibody 3F11 generated against Neisseria gonorrhoeae also binds to this lipooligosaccharide and suggests that the terminal trisaccharide is Gal beta 1----4GlcNAc beta 1----3Gal beta 1----, an epitope found in the glycose moiety of the human erythrocyte glycosphingolipid lactoneotetraglycosylceramide. Mass spectrometric and composition analysis of the lipid A moiety shows that it is similar to the lipid A of Haemophilus influenzae strain I-69 Rd-/b+ proposed by Helander et al. (Helander, I. M., Lindner, B., Brade, H., Altmann, K., Lindberg, A. A., Rietschel, E. T., and Z?hringer, U. (1988) Eur. J. Biochem. 177, 483-492). Electrospray mass spectrometric analysis of the intact O-deacylated lipooligosaccharides gave an average Mr of 2710, and supported an overall structure consisting of the above nonasaccharide linked directly to a diphosphorylated lipid A moiety through the single KDO which is phosphorylated. This structure should provide a framework to investigate the roles of lipooligosaccharides in the host immunochemical response and pathology of H. ducreyi infection, a leading cause of genital ulcer disease.  相似文献   

15.
We describe here the isolation, purification, and structural characterization of a lipid A precursor synthesized under nonpermissive conditions by a mutant of Salmonella typhimurium conditionally defective in the synthesis of the 3-deoxy-D-mannoctulosonate (2-keto-3-deoxyoctonate, KDO) region of the lipopolysaccharide. The precursor was isolated free from lipopolysaccharide, murein, and phospholipids by extraction of delipidated cells with 90% phenol/CHCL3/petroleum ether. The molecule was recovered from the phenol phase after precipitation of lipopolysaccharide with H2O and subsequently purified by DEAE-cellulose chromatography. Structural analyses showed that the lipid A precursor is a phosphorylated glucosamine disaccharide containing one ester and two amide-linked residues of beta-hydroxymyristate. In contrast to lipid A, the precursor disaccharide lacks ester-linked 12:0 and 14:0 fatty acids as well as KDO. The molecule contains 2 phosphate residues both of which were identified as phosphomonoesters by 31P NMR spectroscopy. One of the phosphomonoesters is located in position 1 of the reducing terminal glucosamine residue; the location of the other phosphomonoester was not determined. The structure of the precursor provides strong support for the conclusion that KDO incorporation occurs at an early stage in lipid A biosynthesis prior to the incorporation of ester-linked saturated fatty acids.  相似文献   

16.
Abstract The chemical structure of the lipid A moiety of the lipopolysaccharide of the type strain of Plesiomonas shigelloides was elucidated. It consists of a β-(1 → 6)-linked glucosamine disaccharide carrying phosphate groups at C-1 of the reducing and at C-4' of the non-reducing glucosamine. It contains a total of 6 residues of fatty acids, 2 amide-linked and 4 ester-linked. The amino groups of the backbone disaccharide are N -acylated by substituted 3-hydroxyacyl residues: at the reducing glucosamine by 3-O-(14:0)14:0; and at the non-reducing glucosamine by 3-O-(12:0)14:0.
Two residues of 3-hydroxytetradecanoic acid are linked to C-3 and C-3' of the glucosamine residues; the hydroxy groups of these ester-linked 3-hydroxytetradecanoic acids are unsubstituted. In free lipid A, the hydroxyl groups at C-4 and C-6' are unsubstituted, indicating that the 2-keto-3-deoxyoctonic acid (KDO) is linked to C-6' of the non-reducing glucosamine, as was shown with enterobacterial lipid A. The taxonomical significance of these structural details is discussed.  相似文献   

17.
Oligosaccharides released from the lipooligosaccharides (LOS) of Haemophilus influenzae nontypable strain 2019 by mild acid hydrolysis were fractionated by size exclusion chromatography and analyzed by liquid secondary ion mass spectrometry. The major component of the heterogeneous mixture was found to be a hexasaccharide of Mr 1366, which lost two phosphoethanolamine groups upon treatment with 48% aqueous HF. The dephosphorylated hexasaccharide was purified and shown by tandem mass spectrometry, composition analysis, methylation analysis, and two-dimensional nuclear magnetic resonance studies to be Gal beta 1----4Glc beta 1----(Hep alpha 1----2Hep alpha 1----3) 4Hep alpha 1----5anhydro-KDO, where Hep is L-glycero-D-manno-heptose and KDO is 3-deoxy-D-manno-octulosonic acid. An analogous structure containing authentic KDO was generated from LOS that had been HF-treated prior to acetic acid hydrolysis, suggesting that the reducing terminal anhydro-KDO moiety is produced as an artifact of the hydrolysis procedure by beta-elimination of a phosphate substituent from C-4 of KDO. Mass spectral analyses of O-deacylated LOS and free lipid A confirmed that, in addition to the two phosphoethanolamines on the oligosaccharide and two phosphates on the lipid A, another phosphate group exists on the KDO. This KDO does not appear to be further substituted with additional KDO residues in intact H. influenzae 2019 LOS. The terminal disaccharide epitope, Gal beta 1----4Glc beta 1----, of the hexasaccharide is also present on lactosylceramide, a precursor to human blood group antigens. It is postulated that the presence of this structure on H. influenzae LOS may represent a form of host mimicry by the pathogen.  相似文献   

18.
Inhibition of lipopolysaccharide (LPS) synthesis in Pseudomonas aeruginosa at the stage of incorporation of 3-deoxy-D-manno-octulosonate (KDO) caused accumulation of a lipid A precursor which contained all of the fatty acids present on the lipid A of mature LPS. The enzyme CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) from P. aeruginosa is inhibited by the KDO analog alpha-C-[1,5-anhydro-8-amino-2,7,8-trideoxy-D-manno-octopyranosyl] carboxylate (I), and I is effectively delivered to P. aeruginosa following attachment by amide linkage to the carboxyl terminus of alanylalanine. Intracellular hydrolysis releases the free inhibitor (I) which then inhibits activation of KDO by CMP-KDO synthetase causing accumulation of lipid A precursor and subsequent growth stasis. The major lipid A precursor species accumulated was purified and found to contain glucosamine, phosphate, C12:O, 2OH-C12:O and 3OH-C10:0 (in ester linkage), and 3OH-C12:0 (in amide linkage) in molar ratios of 1:1:0.5:0.5:1:1. Analysis of precursor by fast atom bombardment mass spectroscopy yielded a major ion (M - H)- of mass 1616 and fragments which were consistent with the structure of lipid A from P. aeruginosa. In contrast, Salmonella typhimurium, Escherichia coli, Citrobacter sp., Serratia marcescens, Enterobacter aerogenes, and Enterobacter cloacae all accumulated underacylated lipid A precursors which only contained 3-OH-C14:0, glucosamine, and phosphate. This difference and species-specific patterns of major and minor precursor species show that early steps in the assembly of lipid A are similar, but not identical in enteric and nonenteric Gram-negative bacteria.  相似文献   

19.
Kaustov L  Kababya S  Du S  Baasov T  Gropper S  Shoham Y  Schmidt A 《Biochemistry》2000,39(48):14865-14876
15N?(31)P? REDOR NMR experiments were applied to lyophilized binary complexes of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS), with each of its natural substrates, phosphoenolpyruvate (PEP) and arabinose-5-phsophate (A5P), and with a mechanism-based inhibitor (K(i) = 0.4 microM), directly characterizing the active site basic residues involved in the binding of their carboxylate and phosphate moieties. KDO8PS was labeled uniformly with (15)N or [eta-(15)N(2)]Arg, and the ligands were selectively labeled with (13)C and (15)N. The NMR data established that PEP is bound by KDO8PS via a preserved set of structurally rigid and chemically unique Arg and Lys residues, with 5 A (upper limit) between epsilon-(15)N of this Lys and (31)P of PEP. A5P is bound in its cyclic forms to KDO8PS via a different set of Lys and Arg residues. The two sets arise from adjacent subsites that are capable of independent and sufficiently strong binding. The inhibitor is best characterized as an A5P-based substrate analogue inhibitor of KDO8PS. Five mutants in which highly conserved arginines were replaced with alanines were prepared and kinetically characterized. Our solid-state NMR observations complement the crystallographic structure of KDO8PS, and in combination with the mutagenesis results enable tentative assignment of the NMR-identified active site residues. Lys-138 and Arg-168 located at the most recessed part of the active site cavity are the chemically distinct and structurally rigid residues that bind PEP phosphate; R168A resulted in 0.1% of wild-type activity. Arg-63, exposed at the opening of the active site barrel, is the flexible residue with a generic chemical shift that binds A5P; R63A resulted in complete deactivation. The mechanistic implications of our results are discussed.  相似文献   

20.
Temperature-sensitive mutants of Salmonella typhimurium that are defective in the biosynthesis of 3-deoxy-D-manno-octulosonate are known to accumulate disaccharide precursor(s) of lipid A at 42 degrees C (Rick, P. D., Fung, L. W.-M., Ho, C., and Osborn, M. J. (1977) J. Biol. Chem. 252, 4904-4912). We have devised new methods for purifying this material by chromatography on DEAE-cellulose and silicic acid columns and have fractionated it into eight related anionic components that fall into four sets, as judged by their charge. Substances IA and IB have an apparent net charge of -1, IIA and IIB of -2, IIIA and IIIB of -3, and IVA and IVB of -4. Negative ion fast atom bombardment mass spectrometry reveals that the simplest component is IVA [( M - H]- at m/z 1404). Compound IVA is also the most abundant, representing 30-50% of the accumulated lipids after 3 h at 42 degrees C. Structural studies of IVA, including NMR spectroscopy described in the accompanying paper, reveal that it consists of O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1----6)-2-amino-2-deoxy-alpha - D-glucose, acylated at positions 2, 3, 2', and 3' with beta-hydroxymyristoyl moieties and bearing phosphate groups at positions 1 and 4'. Compound IIIA ([M - H]- at m/z 1527) contains an additional phosphoethanolamine residue, while IIA ([M - H]- m/z 1535) bears an aminodeoxypentose substituent, presumably 4-amino-4-deoxy-L-arabinose. Compound IA ([M - H]- at m/z 1658) bears both a phosphoethanolamine and an aminodeoxypentose. The compounds of the less abundant B series are further derivatized with an ester-linked palmitoyl moiety. Our results demonstrate that these precursors are far more heterogeneous than previously suspected.  相似文献   

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