The Escherichia coli gene encoding the UDP-2,3-diacylglucosamine pyrophosphatase of lipid A biosynthesis

UDP-2,3-diacylglucosamine hydrolase is believed to catalyze the fourth step of lipid A biosynthesis in Escherichia coli. This reaction involves pyrophosphate bond hydrolysis of the precursor UDP-2,3-diacylglucosamine to yield 2,3-diacylglucosamine 1-phosphate and UMP. To identify the gene encoding this hydrolase, E. coli lysates generated with individual lambda clones of the ordered Kohara library were assayed for overexpression of the enzyme. The sequence of lambda clone 157[6E7], promoting overproduction of hydrolase activity, was examined for genes encoding hypothetical proteins of unknown function. The amino acid sequence of one such open reading frame, ybbF, is 50.5% identical to a Haemophilus influenzae hypothetical protein and is also conserved in most other Gram-negative organisms, but is absent in Gram-positives. Cell extracts prepared from cells overexpressing ybbF behind the T7lac promoter have approximately 540 times more hydrolase activity than cells with vector alone. YbbF was purified to approximately 60% homogeneity, and its catalytic properties were examined. Enzymatic activity is maximal at pH 8 and is inhibited by 0.01% (or more) Triton X-100. The apparent K(m) for UDP-2,3-diacylglucosamine is 62 microm. YbbF requires a diacylated substrate and does not cleave CDP-diacylglycerol. (31)P NMR studies of the UMP product generated from UDP-2,3-diacylglucosamine in the presence of 40% H(2)180 show that the enzyme attacks the alpha-phosphate group of the UDP moiety. Because ybbF encodes the specific UDP-2,3-diacylglucosamine hydrolase involved in lipid A biosynthesis, it is now designated lpxH.     

The biosynthesis of gram-negative endotoxin. Formation of lipid A disaccharides from monosaccharide precursors in extracts of Escherichia coli

We have discovered an enzyme in the cytosol of Escherichia coli that generates lipid A disaccharides from monosaccharide precursors by the following route: 2,3-diacyl-GlcN-1-P + UDP-2,3-diacyl-GlcN---- 2,3-diacyl-GlcN (beta, 1----6) 2,3-diacyl-GlcN-1-P + UDP. Previous studies from our laboratory have documented the presence in vivo of the precursors 2,3-diacylglucosamine 1-phosphate (2,3-diacyl-GlcN-1-P) (lipid X of E. coli) and UDP-2,3-diacylglucosamine (UDP-2,3-diacyl-GlcN) (Bulawa, C.E., and Raetz, C.R.H.J. Biol. Chem. 259, 4846-4851). Both substrates are novel glucosamine-derived phospholipids, acylated with beta-hydroxymyristoyl moieties, and they accumulate in E. coli mutants defective in the pgsB gene. Synthetic ADP-, GDP-, and CDP-2,3-diacylglucosamines are inefficient substrates compared to the naturally occurring UDP derivative. The free-acid form of the tetraacyldisaccharide 1-phosphate product (C68H129N2O20P) that is generated in vitro has Mr = 1325.74 as judged by fast atom bombardment mass spectrometry. Mild acid hydrolysis (0.1 M HCl for 30 min at 100 degrees C) liberates greater than 95% of the phosphate moiety as Pi. Detailed analysis by 1H and 13C NMR spectroscopy confirms the presence of a phosphate residue at position 1 of the disaccharide, an alpha-anomeric configuration at the reducing end, and a beta, 1----6 linkage between the two glucosamines. Importantly the disaccharide 1-phosphate synthase is missing in extracts of E. coli strains harboring the pgsB1 mutation, consistent with the massive accumulation of 2,3-diacyl-GlcN-1-P and UDP-2,3-diacyl-GlcN in vivo. The enzymatic reaction reported here represents a major biosynthetic route for the formation of lipid A disaccharides in E. coli and other Gram-negative bacteria. An in vitro system for the biosynthesis of lipid A disaccharides has not been described previously.     

The biosynthesis of gram-negative endotoxin. Identification and function of UDP-2,3-diacylglucosamine in Escherichia coli

Escherichia coli mutants defective in the pgsB gene are phosphatidylglycerol-deficient in certain genetic settings and accumulate novel, glucosamine-derived phospholipids (Nishijima, M., and Raetz, C. R. H. (1979) J. Biol. Chem. 254, 7837-7844). The simplest of these compounds is 2,3-diacylglucosamine 1-phosphate (2,3-diacyl-GlcN-1-P) ("lipid X" of E. coli), in which beta-hydroxymyristoyl moieties are the sole fatty acid substituents (Takayama, K., Qureshi, N., Mascagni, P., Nashed, M. A., Anderson, L., and Raetz, C. R. H. (1983) J. Biol. Chem. 258, 7379-7385). We now report a sensitive radiochemical method for detection of 2,3-diacyl-GlcN-1-P in wild type E. coli and demonstrate that there are about 4000 molecules/cell (0.02% of the total CHCl3-soluble phosphorus). In mutants bearing the pgsB1 lesion, the levels are 100- to 300-fold higher. In addition, we have discovered a novel liponucleotide, UDP-2,3-diacyl-GlcN, that also accumulates in conjunction with the pgsb1 mutation. This material represents 0.005% of the wild type phospholipid and accumulates 50- to 100-fold in the mutant. The identification of UDP-2,3-diacyl-GlcN in E. coli is based on: 1) migration of a minor 32P-labeled lipid from wild type and mutant cells with a UDP-2,3-diacyl-GlCn standard during two-dimensional thin layer chromatography; 2) susceptibility of this 32P-labeled material to cleavage by a liponucleotide-specific pyrophosphatase; and 3) chromatographic identification of [32P]UMP and [32P]2,3-diacyl-GlcN-1-P (lipid X) as the sole products of the enzymatic degradation. As shown in the accompanying article, this novel nucleotide is crucial for biosynthesis of lipid A disaccharides in extracts of E. coli and Salmonella typhimurium.     

Steady-state kinetics and mechanism of LpxD, the N-acyltransferase of lipid A biosynthesis

LpxD catalyzes the third step of lipid A biosynthesis, the (R)-3-hydroxymyristoyl-acyl carrier protein ( R-3-OHC14-ACP)-dependent N-acylation of UDP-3-O-[(R)-3-hydroxymyristoyl]-alpha-D-glucosamine [UDP-3-O-(R-3-OHC14)-GlcN]. We have now overexpressed and purified Escherichia coli LpxD to homogeneity. Steady-state kinetics suggest a compulsory ordered mechanism in which R-3-OHC14-ACP binds prior to UDP-3-O-(R-3-OHC14)-GlcN. The product, UDP-2,3-diacylglucosamine, dissociates prior to ACP; the latter is a competitive inhibitor against R-3-OHC14-ACP and a noncompetitive inhibitor against UDP-3-O-(R-3-OHC14)-GlcN. UDP-2-N-[(R)-3-Hydroxymyristoyl]-alpha-D-glucosamine, obtained by mild base hydrolysis of UDP-2,3-diacylglucosamine, is a noncompetitive inhibitor against both substrates. Synthetic (R)-3-hydroxylauroyl-methylphosphopantetheine is an uncompetitive inhibitor against R-3-OHC14-ACP and a competitive inhibitor against UDP-3-O-(R-3-OHC14)-GlcN, but (R)-3-hydroxylauroyl-methylphosphopantetheine is also a very poor substrate. A compulsory ordered mechanism is consistent with the fact that R-3-OHC14-ACP has a high binding affinity for free LpxD whereas UDP-3-O-(R-3-OHC14)-GlcN does not. Divalent cations inhibit R-3-OHC14-ACP-dependent acylation but not (R)-3-hydroxylauroyl-methylphosphopantetheine-dependent acylation, indicating that the acidic recognition helix of R-3-OHC14-ACP contributes to binding. The F41A mutation increases the K(M) for UDP-3-O-(R-3-OHC14)-GlcN 30-fold, consistent with aromatic stacking of the corresponding F43 side chain against the uracil moiety of bound UDP-GlcNAc in the X-ray structure of Chlamydia trachomatis LpxD. Mutagenesis implicates E. coli H239 but excludes H276 as the catalytic base, and neither residue is likely to stabilize the oxyanion intermediate.     

The UDP-diacylglucosamine Pyrophosphohydrolase LpxH in Lipid A Biosynthesis Utilizes Mn2+ Cluster for Catalysis

In Escherichia coli and the majority of β- and γ-proteobacteria, the fourth step of lipid A biosynthesis, i.e. cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN, is carried out by LpxH. LpxH has been previously suggested to contain signature motifs found in the calcineurin-like phosphoesterase (CLP) family of metalloenzymes; however, it cleaves a pyrophosphate bond instead of a phosphoester bond, and its substrate contains nucleoside diphosphate moieties more common to the Nudix family rather than to the CLP family. Furthermore, the extent of biochemical data fails to demonstrate a significant level of metal activation in enzymatic assays, which is inconsistent with the behavior of a metalloenzyme. Here, we report cloning, purification, and detailed enzymatic characterization of Haemophilus influenzae LpxH (HiLpxH). HiLpxH shows over 600-fold stimulation of hydrolase activity in the presence of Mn²⁺. EPR studies reveal the presence of a Mn²⁺ cluster in LpxH. Finally, point mutants of residues in the conserved metal-binding motifs of the CLP family greatly inhibit HiLpxH activity, highlighting their importance in enzyme function. Contrary to previous analyses of LpxH, we find HiLpxH does not obey surface dilution kinetics. Overall, our work unambiguously establishes LpxH as a calcineurin-like phosphoesterase containing a Mn²⁺ cluster coordinated by conserved residues. These results set the scene for further structural investigation of the enzyme and for design of novel antibiotics targeting lipid A biosynthesis.     

Gene expression and biochemical characterization of Azotobacter vinelandii cyclophilins and Protein Interaction Studies of the cytoplasmic isoform with dnaK and lpxH

The soil nitrogen-fixing bacterium Azotobacter vinelandii possesses two cyclophilins, comprising putative cytoplasmic and periplasmic isoforms, designated as AvPPIB and AvPPIA, respectively. Both recombinant cyclophilins have been purified and their peptidyl-prolyl cis/trans isomerase activity against Suc-Ala-Xaa-Pro-Phe-pNA synthetic peptides has been characterized. The substrate specificity of both cyclophilins is typical for bacterial cyclophilins, with Suc-Ala-Ala-Pro-Phe-pNA being the most rapidly catalyzed substrate. The cytoplasmic cyclophilin also displays a chaperone function in the citrate synthase thermal aggregation assay. Using real-time quantitative RT-PCR, we demonstrate that AvppiB is expressed under various physiological and growth conditions, mainly upregulated by acetate and downregulated by the stationary growth state, while AvppiA shows a tendency for downregulation under the tested conditions. Further, we identified chaperone protein dnaK and UDP-2, 3-diacylglucosamine hydrolase lpxH as probable interacting partners of AvPPIB and we demonstrate their physical interaction by coexpression studies. An increase in AvPPIB PPIase activity in the presence of AvdnaK and a decrease in the presence of AvlpxH further confirms each interaction. However, the PPIase activity does not seem to be essential for those interactions since AvPPIB active site mutants still interact with dnaK and lpxH, while their minor PPIase activity cannot be modulated by the interaction.     

Biosynthesis of lipid A in Escherichia coli: identification of UDP-3-O-[(R)-3-hydroxymyristoyl]-alpha-D-glucosamine as a precursor of UDP-N2,O3-bis[(R)-3-hydroxymyristoyl]-alpha-D-glucosamine

The lipid A disaccharide of the Escherichia coli envelope is synthesized from the two fatty acylated glucosamine derivatives UDP-N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D- glucosamine (UDP-2,3-diacyl-GlcN) and N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D-glucosamine 1-phosphate (2,3-diacyl-GlcN-1-P) [Ray, B. L., Painter, G., & Raetz, C. R. H. (1984) J. Biol. Chem. 259, 4852-4859]. We have previously shown that UDP-2,3-diacyl-GlcN is generated in extracts of E. coli by fatty acylation of UDP-GlcNAc, giving UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc as the first intermediate, which is rapidly converted to UDP-2,3-diacyl-GlcN [Anderson, M. S., Bulawa, C. E., & Raetz, C. R. H. (1985) J. Biol. Chem. 260, 15536-15541; Anderson, M. S., & Raetz, C. R. H. (1987) J. Biol. Chem. 262, 5159-5169]. We now demonstrate a novel enzyme in the cytoplasmic fraction of E. coli, capable of deacetylating UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc to form UDP-3-O-[(R)-3-hydroxymyristoyl]glucosamine. The covalent structure of the previously undescribed UDP-3-O-[(R)-3-hydroxymyristoyl] glucosamine intermediate was established by 1H NMR spectroscopy and fast atom bombardment mass spectrometry. This material can be made to accumulate in E. coli extracts upon incubation of UDP-3-O-[(R)-3- hydroxymyristoyl]-GlcNAc in the absence of the fatty acyl donor [(R)-3-hydroxymyristoyl]-acyl carrier protein. However, addition of the isolated deacetylation product [UDP-3-O-[(R)-3-hydroxymyristoyl] glucosamine] back to membrane-free extracts of E. coli in the presence of [(R)-3-hydroxymyristoyl]-acyl carrier protein results in rapid conversion of this compound into the more hydrophobic products UDP-2,3-diacyl-GlcN, 2,3-diacyl-GlcN-1-P, and O-[2-amino-2-deoxy-N2,O3- bis[(R)-3-hydroxytetradecanoyl]-beta-D-glucopyranosyl]-(1----6)-2-amino- 2-deoxy-N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D- glucopyranose 1-phosphate (tetra-acyldisaccharide-1-P), demonstrating its competency as a precursor. In vitro incubations using [acetyl-3H]UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc confirmed release of the acetyl moiety in this system as acetate, not as some other acetyl derivative. The deacetylation reaction was inhibited by 1 mM N-ethylmaleimide, while the subsequent N-acylation reaction was not. Our observations provide strong evidence that UDP-3-O-[(R)-3-hydroxymyristoyl]glucosamine is a true intermediate in the biosynthesis of UDP-2,3-diacyl-GlcN and lipid A.     

The biosynthesis of gram-negative endotoxin. A novel kinase in Escherichia coli membranes that incorporates the 4'-phosphate of lipid A

Extracts of Escherichia coli contain an enzyme that generates the beta,1----6 linkage of lipid A from fatty-acylated monosaccharide precursors, according to the reaction: 2,3-diacyl-GlcN-1-P + UDP-2,3-diacyl-GlcN----2,3-diacyl-GlcN (beta, 1----6)2,3-diacyl-GlcN-1-P + UDP (Ray, B. L., Painter, G., and Raetz, C. R. H. (1984) J. Biol. Chem. 259, 4852-4859). We now describe a membrane-bound kinase that phosphorylates the 4'-position of the above tetraacyldisaccharide 1-phosphate product. The lipid A 4'-kinase is distinct from the diglyceride kinase of E. coli. When crude membrane preparations are employed, several nucleoside triphosphates are able to support the phosphorylation of the tetraacyldisaccharide 1-phosphate, but ATP is the most efficient. The 4'-kinase requires Mg2+ and is stimulated by phospholipids, especially cardiolipin. Under optimal conditions the specific activity in crude extracts is 0.5 nmol/min/mg. The enzyme is rapidly inactivated by preincubation in the presence of detergents, such as Nonidet P-40 or octylglucoside, but phosphoenolpyruvate and glycerol stabilize the enzyme. The product generated in vitro has been characterized by fast atom bombardment mass spectrometry and by 1H and 31P NMR spectroscopy. Those analyses confirm that the 4' hydroxyl is the site of phosphorylation. The 4'-kinase reported here is likely to represent a key step in the de novo biosynthesis of lipid A.     

Purification and properties of lipid A disaccharide synthase of Escherichia coli

Lipid A disaccharide synthase of Escherichia coli catalyzes the reaction 2,3-diacyl-GlcN-1-P + UDP-2,3-diacyl-GlcN----2',3'-diacyl-GlcN (beta,1'----6)2,3-diacyl-GlcN-1-P + UDP (Ray, B. L., Painter, G., and Raetz, C. R. H. (1984) J. Biol. Chem. 259, 4852-4859). Using a strain that overproduces the enzyme about 200-fold we have devised a simple purification to near homogeneity, utilizing two types of dye-ligand resins and heparin-agarose. The overall purification starting with membrane-free extracts was 54-fold (16,000-fold relative to wild-type extracts) with a 31% yield. The subunit molecular mass determined by sodium dodecyl sulfate gel electrophoresis is approximately 42,000 daltons, and the native enzyme appears to be a dimer. The amino-terminal sequence is (X)-(Thr)-Glu-Gln-(X)-Pro-Leu-Thr-Ie-Ala..., consistent with the results predicted from the DNA sequence, Met-Thr-Glu-Gln-Arg-Pro-Leu-Thr-Ile-Ala.... The purified enzyme displays a strong kinetic preference for sugar substrates bearing two fatty acyl moieties, but it is, nevertheless, very useful for the semisynthetic preparation of many lipid A analogs. Gel filtration studies demonstrate that the natural substrates (2,3-diacyl-GlcN-1-P and UDP-2,3-diacyl-GlcN) form micelles (n approximately equal to 300), rather than bilayers, under conditions used to assay the enzyme. Unlike most enzymes of glycerophospholipid synthesis, the lipid A disaccharide synthase does not require the presence of a detergent for catalytic activity. At 1 mM UDP-2,3-diacyl-GlcN the Vmax and Km values for 2,3-diacyl-GlcN-1-P are 14,028 +/- 513 nmol/min/mg and 0.27 +/- 0.02 mM. When 2,3-diacyl-GlcN-1-P is maintained at 1 mM, they are 12,368 +/- 472 nmol/min/mg and 0.11 +/- 0.01 mM for UDP-2,3-diacyl-GlcN.     

Enzymatic characterization of 5-methylthioribulose-1-phosphate dehydratase of the methionine salvage pathway in Bacillus subtilis

5-Methylthioribulose-1-phosphate (MTRu-1-P) dehydratase catalyzes the reaction from MTRu-1-P to 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) in the methionine salvage pathway in Bacillus subtilis. The properties of this enzyme remain to be determined. We characterized these properties using a recombinant protein. The enzyme, with a molecular mass of 90 kDa, was composed of four subunits. The K(m) and V(max) of the enzyme were 8.9 microM and 42.7 micromole min(-1) mg protein(-1) at 25 degrees C respectively. Maximum activity was observed at pH 7.5 to 8.5 and 40 degrees C. The activation energy of the reaction from MTRu-1-P to DK-MTP-1-P was 63.5 kJ mol(-1). The reaction product DK-MTP-1-P was labile, and decomposed at a rate constant of 0.048 s(-1) to an unknown compound that was not utilized by DK-MTP-1-P enolase, the enzyme catalyzing the next step. The function of this enzyme in the pathway is discussed.     

Synthesis, characterization and properties of uridine 5′-(α-d-apio-d-furanosyl pyrophosphate)

1. A method was developed for synthesizing UDP-apiose [uridine 5'-(alpha-d-apio-d-furanosyl pyrophosphate)] from UDP-glucuronic acid [uridine 5'-(alpha-d-glucopyranosyluronic acid pyrophosphate)] in 62% yield with the enzyme UDP-glucuronic acid cyclase. 2. UDP-apiose had the same mobility as uridine 5'-(alpha-d-xylopyranosyl pyrophosphate) when chromatographed on paper and when subjected to paper electrophoresis at pH5.8. When [(3)H]UDP-[U-(14)C]glucuronic acid was used as the substrate for UDP-glucuronic acid cyclase, the (3)H/(14)C ratio in the reaction product was that expected if d-apiose remained attached to the uridine. In separate experiments doubly labelled reaction product was: (a) hydrolysed at pH2 and 100 degrees C for 15min; (b) degraded at pH8.0 and 100 degrees C for 3min; (c) used as a substrate in the enzymic synthesis of [(14)C]apiin. In each type of experiment the reaction products were isolated and identified and were found to be those expected if [(3)H]UDP-[U-(14)C]apiose was the starting compound. 3. Chemical characterization established that the product containing d-[U-(14)C]apiose and phosphate formed on alkaline degradation of UDP-[U-(14)C]apiose was alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate. 4. Chemical characterization also established that the product containing d-[U-(14)C]apiose and phosphate formed on acid hydrolysis of alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate was d-[U-(14)C]apiose 2-phosphate. 5. The half-life periods for the degradation of UDP-[U-(14)C]apiose to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP at pH8.0 and 80 degrees C, at pH8.0 and 25 degrees C and at pH8.0 and 4 degrees C were 31.6s, 97.2min and 16.5h respectively. The half-life period for the hydrolysis of UDP-[U-(14)C]-apiose to d-[U-(14)C]apiose and UDP at pH3.0 and 40 degrees C was 4.67min. After 20 days at pH6.2-6.6 and 4 degrees C, 17% of the starting UDP-[U-(14)C]apiose was degraded to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP and 23% was hydrolysed to d-[U-(14)C]apiose and UDP. After 120 days at pH6.4 and -20 degrees C 2% of the starting UDP-[U-(14)C]apiose was degraded and 4% was hydrolysed.     

Biosynthesis of riboflavin in archaea studies on the mechanism of 3,4-dihydroxy-2-butanone-4-phosphate synthase of Methanococcus jannaschii

The hypothetical protein predicted by the open reading frame MJ0055 of Methanococcus jannaschii was expressed in a recombinant Escherichia coli strain under the control of a synthetic gene optimized for translation in an eubacterial host. The recombinant protein catalyzes the formation of the riboflavin precursor 3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5-phosphate at a rate of 174 nmol mg(-1) min(-1) at 37 degrees C. The homodimeric 51.6-kDa protein requires divalent metal ions, preferentially magnesium, for activity. The reaction involves an intramolecular skeletal rearrangement as shown by (13)C NMR spectroscopy using [U-(13)C(5)]ribulose 5-phosphate as substrate. A cluster of charged amino acid residues comprising arginine 25, glutamates 26 and 28, and aspartates 21 and 30 is essential for catalytic activity. Histidine 164 and glutamate 185 were also shown to be essential for catalytic activity.     

Biosynthesis of lipid A precursors in Escherichia coli. A membrane-bound enzyme that transfers a palmitoyl residue from a glycerophospholipid to lipid X

Certain phosphatidylglycerol-deficient mutants of Escherichia coli accumulate two fatty acylated monosaccharides related to lipid A biosynthesis that have been identified as 2,3-diacylglucosamine 1-phosphate (lipid X) and triacylglucosamine 1-phosphate (lipid Y) (Raetz, C. R. H. (1984) Rev. Infect. Dis. 6, 463-472). Lipid Y has the same structure as lipid X, except that it bears an additional palmitoyl moiety, esterified to the 3-OH of the N-linked R-3-hydroxymyristoyl residue. We now describe a membrane-associated system for the enzymatic conversion of lipid X to lipid Y. Removal of glycerophospholipids form such membranes by washing with cold ethanol abolishes the activity. The system can be reactivated by the addition of exogenous phospholipids dispersed as mixed micelles with Triton X-100. When reconstituted in this manner, the formation of lipid Y is strictly dependent upon a glycerophospholipid donor bearing a palmitoyl residue in the sn-1 position. The enzyme system does not utilize palmitoyl coenzyme A or palmitoyl acyl carrier protein. It does not catalyze efficient transfer of fatty acids differing from palmitate by only one carbon atom. In contrast, the enzyme has relatively little specificity for the polar headgroup of the phospholipid donor, and it also appears to utilize a disaccharide precursor of lipid A as an alternative palmitoyl acceptor. Since the in vitro synthesis of lipid Y proceeds with a high yield, we have isolated the product and verified its structure by 1H NMR spectroscopy and mass spectrometry. The transesterification reaction that converts lipid X to lipid Y may be a model for the enzymatic synthesis of other acyloxyacyl structures, known to occur in mature lipid A.     

The biosynthesis of gram-negative endotoxin. Formation of lipid A precursors from UDP-GlcNAc in extracts of Escherichia coli

The Gram-negative bacterium Escherichia coli has previously been shown to utilize two unique glucosamine (GlcN)-derived phospholipids in the biosynthesis of lipid A disaccharides (Bulawa, C.E., and Raetz, C. R.H. (1984) J. Biol. Chem. 259, 4846-4851; Ray, B. L., Painter, G.L., and Raetz, C.R.H. (1984) J. Biol. Chem. 259, 4852-4859. We now present evidence that these compounds, UDP-2,3-diacyl-GlcN and 2,3-diacyl-GlcN-1-phosphate (2,3-diacyl-GlcN-1-P), are generated in extracts of E. coli by fatty acylation of UDP-GlcNAc. The initial reaction is an O-acylation of the glucosamine ring, presumably of the 3-OH group, with (R)-beta-hydroxymyristate, followed by removal of the acetyl moiety, and further fatty acylation of the N atom with (R)-beta-hydroxymyristate to yield UDP-2,3-diacyl-GlcN. Hydrolysis of the pyrophosphate bridge in this molecule gives 2,3-diacyl-GlcN-1-P + UMP. In vivo pulse labeling with 32Pi supports this postulated pathway, since UDP-2,3-diacyl-GlcN is labeled prior to 2,3-diacyl-GlcN-1-P. UDP-glucosamine is inactive as a substrate in the initial acylation reaction. These acylations show an absolute specificity for fatty acyl moieties activated with acyl carrier protein. No reaction is detected with fatty acyl-CoA or free fatty acid. The fatty acylation of sugar nucleotides has not been reported previously in E. coli or any other organism.     

Pulmonary pressor responses in sheep to chemically defined precursors of E. coli endotoxin

The toxicity of various monosaccharide and disaccharide endotoxin precursors has now been studied in sheep. We measured the early pulmonary arterial pressure responses after injections of the monosaccharides lipid X (2,3-diacylglucosamine 1-phosphate) and MAGP (2-monoacylglucosamine 1-phosphate), of the tetraacyl disaccharide diphosphate precursor of lipid A, IV-A (Federation Proc. 43: 1567, 1984), and of Escherichia coli bacterial endotoxin (lipopolysaccharide). We also measured the response of lipid X after prior administration of indomethacin and MAGP. Lipid X, at a total cumulative dose of 40 micrograms/kg, produced an immediate, but transient dose-dependent pulmonary arterial vasoconstrictive response. MAGP, at a total dose of 40 micrograms/kg, had no pulmonary pressure activity but did increase extravascular lung water and produce some histological changes in the lung. Disaccharide precursor IV-A, at a total dose of 40 micrograms/kg, produced an immediate dose-dependent pulmonary arterial vasoconstrictive response that was prolonged for greater than 2 h. E. coli endotoxin caused a delayed (15-min) increase in the pulmonary arterial pressure but one that also persisted for greater than 2 h. Prior administration of indomethacin blocked the pulmonary pressor activity of lipid X, whereas prior administration of MAGP increased both the magnitude and the duration of the pulmonary pressure response of lipid X. We conclude that the initial pulmonary hypertension seen after lipid X injection may involve cyclooxygenase-dependent formation of prostaglandins and that the genesis of this pulmonary pressor activity is at least in part dependent on the ester-linked hydroxymyristoyl moiety at position 3 of the lipid X molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of phosphatidylinositol 4,5-bisphosphate phospholipase C activity by phosphatidic acid

Phosphatidic acid was a potent activator of the phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) phospholipase C activity associated with human platelet membranes. Lysophosphatidic acid was half as active as phosphatidic acid, and shortening the fatty acid chain reduced the effectiveness of the corresponding phosphatidic acid. Compounds lacking either the phosphate group (diacylglycerol or phorbol ester) or the fatty acid (glycerol phosphate) were not activators. When the negative charge was contributed by a carboxyl group (fatty acid or phosphatidylserine), stimulation of phospholipase C was weak but detectable. Structural analogs of phosphatidic acid (lipopolysaccharide, lipid A, and 2,3-diacylglucosamine 1-phosphate) were less effective but also enhanced PtdIns-P2 hydrolysis. Phosphatidic acid potentiated the activation of phospholipase C by alpha-thrombin, chelators, and guanine nucleotides. Phosphatidylinositol 4-phosphate and PtdIns-P2 were also effective activators of PtdIns-P2 degradation. Other phospholipids were without effect. The production of inositol 1,4,5-trisphosphate and diacylglycerol via the activation of phospholipase C provides a rationale for the cellular responses evoked by phosphatidic acid and the ability of this phospholipid to potentiate and initiate hormonal responses.     

Activation of cyclic nucleotide phosphodiesterase by a monosaccharide precursor of Escherichia coli lipid A

The E. coli lipid A precursor lipid X (N2,O3-diacylglucosamine 1-phosphate) activates calmodulin-dependent cyclic nucleotide phosphodiesterase in a noncooperative, calcium-independent manner by increasing its Vmax and decreasing its Km for substrate. The glycolipid produces half-maximal activation at 11 micrograms/ml and does not further enhance activation by calcium-calmodulin. Lipid X activation of phosphodiesterase requires the presence of the O3-linked hydroxymyristoyl residue. These findings suggest that lipid X could produce some of its biological effects by modulating intracellular cAMP levels.     

The activation of protein kinase C by biologically active lipid moieties of lipopolysaccharide

The monosaccharide lipid A precursor, N2,O3-diacylglucosamine 1-phosphate (Escherichia coli lipid X), has been shown previously to be a potent B-lymphocyte mitogen. We now report that lipid X interacts with macrophages, stimulating turnover of phosphatidylinositol, deacylation of phospholipids, and release of arachidonic acid. In addition, the monosaccharide lipid X, the incomplete lipid A disaccharides found in KDO-deficient mutants, and crude free lipid A by itself activate protein kinase C isolated from RAW 264.7 macrophages. This activation is augmented by diglyceride, a product of phosphatidylinositol turnover. Like the lipid X-induced mitogenesis of B-lymphocytes, lipid X activation of macrophages and the cell-free activation of protein kinase by lipid X require the presence of the O-linked hydroxymyristoyl residue at position 3. We suggest, therefore, that some of the biological effects of lipid A may be mediated by its interaction with protein kinase C.     

Biosynthesis of the wall acidic polysaccharide in Bacillus cereus AHU 1356

Biosynthetic studies on an acidic polysaccharide, comprising galactose, rhamnose, N-acetylglucosamine and sn-glycerol 1-phosphate, were carried out with a membrane system obtained from Bacillus cereus AHU 1356. Incubation of the membranes with UDP-[14C]Gal, TDP-[14C]Rha and UDP-[14C]GlcNAc resulted in the formation of four or more labeled-sugar-linked lipids and a labeled polysaccharide. Data on structural analysis of the sugar moieties released from the glycolipids, together with results of enzymatic conversion of [14C]galactose-linked lipid and [14C]Rha-Gal-linked lipid to higher-oligosaccharide-linked lipids and polysaccharide, led to the conclusion that the acidic polysaccharide is probably synthesized through the following pathway: (sequence in text) The glycerophosphate residues seem to be derived from phosphatidylglycerol.     

Gene expression and molecular characterization of a thermostable trehalose phosphorylase fromThermoanaerobacter tengcongensis

Trehalose is a non-reducing disaccharide of glucosewidely distributed in microorganisms, plants and in-sects. It usually functions as a compatible solute in thestabilization of biological structures under several en-vironment stresses[1,2]. Trehalose has proved to be anactive stabilizer of enzymes, proteins, biomasses, pharmaceutical preparations and even organs fortransplantation. Thus much attention has been paid tothe synthesis pathway of trehalose and the develop-ment of novel and economic…