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1.
Partial Purification and Properties of l-Glutamine d-Fructose 6-Phosphate Amidotransferase from Phaseolus aureus 总被引:1,自引:1,他引:0
l-Glutamine d-fructose 6-phosphate amidotransferase (EC 2.6.1.16) was extracted and purified 600-fold by acetone fractionation and diethylaminoethyl cellulose column chromatography from mung bean seeds (Phaseolus aureus). The partially purified enzyme was highly specific for l-glutamine as an amide nitrogen donor, and l-asparagine could not replace it. The enzyme showed a pH optimum in the range of 6.2 to 6.7 in phosphate buffer. Km values of 3.8 mm and 0.5 mm were obtained for d-fructose 6-phosphate and l-glutamine, respectively. The enzyme was competitively inhibited with respect to d-fructose 6-phosphate by uridine diphosphate-N-acetyl-d-glucosamine which had a Ki value of 13 μm. Upon removal of l-glutamine and its replacement by d-fructose 6-phosphate and storage over liquid nitrogen, the enzyme was completely desensitized to inhibition by uridine diphosphate-N-acetyl-d-glucosamine. This indicates that the inhibitor site is distinct from the catalytic site and that uridine diphosphate-N-acetyl-d-glucosamine acts as a feedback inhibitor of the enzyme. 相似文献
2.
Takanori Nihira Erika Suzuki Motomitsu Kitaoka Mamoru Nishimoto Ken'ichi Ohtsubo Hiroyuki Nakai 《The Journal of biological chemistry》2013,288(38):27366-27374
A gene cluster involved in N-glycan metabolism was identified in the genome of Bacteroides thetaiotaomicron VPI-5482. This gene cluster encodes a major facilitator superfamily transporter, a starch utilization system-like transporter consisting of a TonB-dependent oligosaccharide transporter and an outer membrane lipoprotein, four glycoside hydrolases (α-mannosidase, β-N-acetylhexosaminidase, exo-α-sialidase, and endo-β-N-acetylglucosaminidase), and a phosphorylase (BT1033) with unknown function. It was demonstrated that BT1033 catalyzed the reversible phosphorolysis of β-1,4-d-mannosyl-N-acetyl-d-glucosamine in a typical sequential Bi Bi mechanism. These results indicate that BT1033 plays a crucial role as a key enzyme in the N-glycan catabolism where β-1,4-d-mannosyl-N-acetyl-d-glucosamine is liberated from N-glycans by sequential glycoside hydrolase-catalyzed reactions, transported into the cell, and intracellularly converted into α-d-mannose 1-phosphate and N-acetyl-d-glucosamine. In addition, intestinal anaerobic bacteria such as Bacteroides fragilis, Bacteroides helcogenes, Bacteroides salanitronis, Bacteroides vulgatus, Prevotella denticola, Prevotella dentalis, Prevotella melaninogenica, Parabacteroides distasonis, and Alistipes finegoldii were also suggested to possess the similar metabolic pathway for N-glycans. A notable feature of the new metabolic pathway for N-glycans is the more efficient use of ATP-stored energy, in comparison with the conventional pathway where β-mannosidase and ATP-dependent hexokinase participate, because it is possible to directly phosphorylate the d-mannose residue of β-1,4-d-mannosyl-N-acetyl-d-glucosamine to enter glycolysis. This is the first report of a metabolic pathway for N-glycans that includes a phosphorylase. We propose 4-O-β-d-mannopyranosyl-N-acetyl-d-glucosamine:phosphate α-d-mannosyltransferase as the systematic name and β-1,4-d-mannosyl-N-acetyl-d-glucosamine phosphorylase as the short name for BT1033. 相似文献
3.
The uptake of d-galactose was studied in detached fenugreek (Trigonella foenum-graecum L.) cotyledons. Uptake kinetics and treatment with p-chloromercury-benzenesulfonic acid indicated that at low concentrations d-galactose was taken up by a carrier. At higher concentrations a diffusion-like component existed. Proton flux and pH studies, treatment with α-naphthaleneacetic acid, and uptake experiments under water stress conditions suggested that d-galactose was not taken up via H+ contransport. However, d-galactose uptake was under metabolic control. Uptake kinetics under water stress conditions suggested that moderate water stress either increased the Km of the carrier or decreased the Vmax. However, prolonged stress transformed the carrier-mediated uptake into a diffusion uptake transport. The uptake of d-galactose by fenugreek cotyledons was very low before and just after germination, was maximum after 35 hours imbibition, and started decreasing thereafter. The different uptake rates of d-galactose with imbibition times were attributed to the operation of the carrier. At low uptake rates the carrier did not operate. Treatment with cycloheximide suggested that the carrier was synthesized de novo just after germination and stopped operating when all galactomannan hydrolysis was over. Results were discussed in the context of control of endosperm galactomannan hydrolysis by the cotyledons of fenugreek embryo. 相似文献
4.
The isolation of oligosaccharides from the cell-wall polysaccharide of Lactobacillus casei, serological group C 总被引:3,自引:3,他引:0
1. A number of disaccharides and oligosaccharides have been isolated from the products of mild acid hydrolysis of the specific substance from Lactobacillus casei, serological group C. 2. The major disaccharide is O-β-d-glucopyranosyl-(1→3)-N-acetyl- d-galactosamine (B4) and evidence is presented for the structure of a tetrasaccharide composed of O-β-d-glucopyranosyl-(1→6)-d-galactose (B1) joined through its reducing end group to B4. 3. Disaccharide B1 is also a component of a trisaccharide O-β-d-glucopyranosyl-(1→6)-O-β- d-galactopyranosyl-(1→6)-N-acetyl-d-glucosamine (A7). 4. A number of other oligosaccharides have been shown to be related structurally. 5. The ability of certain of the oligosaccharides to inhibit the precipitin reaction has been studied. The disaccharide B1 is more effective as an inhibitor than gentiobiose and the trisaccharide A7 is considerably more effective than B1. 6. These results have been compared with those obtained previously for the composition of the cell wall. 相似文献
5.
Pathway of Uridine Diphosphate N-Acetyl-d-Glucosamine Biosynthesis in Phaseolus aureus 总被引:3,自引:3,他引:0
Studies with extracts obtained from mung beans (Phaseolus aureus) showed that UDP-N-acetyl d-glucosamine is formed from d-fructose 6-phosphate by a series of the following enzymic reactions: [Formula: see text] 相似文献
6.
The incorporation of labelled amino sugars by Bacillus subtilis 总被引:1,自引:1,他引:0
1. Glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] of Bacillus subtilis has been partially purified. Its Km is 3·0mm. 2. Extracts of B. subtilis contain N-acetylglucosamine 6-phosphate deacetylase (Km 1·4mm), glucosamine 1-phosphate acetylase and amino sugar kinases (EC 2.7.1.8 and 2.7.1.9). 3. Glucosamine 6-phosphate synthetase (l-glutamine–d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) is repressed by growth of B. subtilis in the presence of glucosamine, N-acetylglucosamine, N-propionylglucosamine or N-formylglucosamine. Glucosamine 6-phosphate deaminase and N-acetylglucosamine 6-phosphate deacetylase are induced by N-acetylglucosamine. Amino sugar kinases are induced by glucose, glucosamine and N-acetylglucosamine. The synthesis of glucosamine 1-phosphate acetylase is unaffected by amino sugars. 4. Glucose in the growth medium prevents the induction of glucosamine 6-phosphate deaminase and of N-acetylglucosamine 6-phosphate deacetylase caused by N-acetylglucosamine; glucose also alleviates the repression of glucosamine 6-phosphate synthetase caused by amino sugars. 5. Glucosamine 6-phosphate deaminase increases in bacteria incubated beyond the exponential phase of growth. This increase is prevented by glucose. 相似文献
7.
1. Glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] of Bacillus subtilis has been partially purified. Its Km is 3·0mm. 2. Extracts of B. subtilis contain N-acetylglucosamine 6-phosphate deacetylase (Km 1·4mm), glucosamine 1-phosphate acetylase and amino sugar kinases (EC 2.7.1.8 and 2.7.1.9). 3. Glucosamine 6-phosphate synthetase (l-glutamine–d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) is repressed by growth of B. subtilis in the presence of glucosamine, N-acetylglucosamine, N-propionylglucosamine or N-formylglucosamine. Glucosamine 6-phosphate deaminase and N-acetylglucosamine 6-phosphate deacetylase are induced by N-acetylglucosamine. Amino sugar kinases are induced by glucose, glucosamine and N-acetylglucosamine. The synthesis of glucosamine 1-phosphate acetylase is unaffected by amino sugars. 4. Glucose in the growth medium prevents the induction of glucosamine 6-phosphate deaminase and of N-acetylglucosamine 6-phosphate deacetylase caused by N-acetylglucosamine; glucose also alleviates the repression of glucosamine 6-phosphate synthetase caused by amino sugars. 5. Glucosamine 6-phosphate deaminase increases in bacteria incubated beyond the exponential phase of growth. This increase is prevented by glucose. 相似文献
8.
Lectins extracted from corn (Zea mays L.) kernel with Tris-HCl buffer pH 7.5 were isolated from the crude extract by affinity chromatography on Sepharose 6B-N-acetyl-d-galactosamine and Sepharose 6B-methylα-d-mannoside, and also by lectin affinity chromatography using concanavalin A and Lens culinaris lectin as ligands. According to preferential monosaccharide specificity, salt-soluble lectins of corn seed comprise at least two distinct types: N-acetyl-d-galactosamine-interactive and mannose-interactive lectins. The extracted lectins are unstable, with a tendency to form aggregates during storage. 相似文献
9.
Kimran Hayer Malcolm Stratford David B. Archer 《Applied and environmental microbiology》2013,79(22):6924-6931
The asexual spores (conidia) of Aspergillus niger germinate to produce hyphae under appropriate conditions. Germination is initiated by conidial swelling and mobilization of internal carbon and energy stores, followed by polarization and emergence of a hyphal germ tube. The effects of different pyranose sugars, all analogues of d-glucose, on the germination of A. niger conidia were explored, and we define germination as the transition from a dormant conidium into a germling. Within germination, we distinguish two distinct stages, the initial swelling of the conidium and subsequent polarized growth. The stage of conidial swelling requires a germination trigger, which we define as a compound that is sensed by the conidium and which leads to catabolism of d-trehalose and isotropic growth. Sugars that triggered germination and outgrowth included d-glucose, d-mannose, and d-xylose. Sugars that triggered germination but did not support subsequent outgrowth included d-tagatose, d-lyxose, and 2-deoxy-d-glucose. Nontriggering sugars included d-galactose, l-glucose, and d-arabinose. Certain nontriggering sugars, including d-galactose, supported outgrowth if added in the presence of a complementary triggering sugar. This division of functions indicates that sugars are involved in two separate events in germination, triggering and subsequent outgrowth, and the structural features of sugars that support each, both, or none of these events are discussed. We also present data on the uptake of sugars during the germination process and discuss possible mechanisms of triggering in the absence of apparent sugar uptake during the initial swelling of conidia. 相似文献
10.
By the use of 1mm-iodoacetate to inhibit glycolysis in guinea-pig cerebral tissue slices, the kinetics of the uptake of monosaccharides on transfer of tissue from 0° to 37° were studied. d-Ribose, d-galactose, d-mannose, l-sorbose, and d-fructose showed diffusion kinetics, whereas 2-deoxy-d-glucose, d-glucose, d-arabinose and d-xylose showed saturation kinetics. 相似文献
11.
Inositol Metabolism in Plants. V. Conversion of Myo-inositol to Uronic Acid and Pentose Units of Acidic Polysaccharides in Root-tips of Zea mays 总被引:8,自引:7,他引:1
The metabolism of myo-inositol-2-14C, d-glucuronate-1-14C, d-glucuronate-6-14C, and l-methionine-methyl-14C to cell wall polysaccharides was investigated in excised root-tips of 3 day old Zea mays seedlings. From myo-inositol, about one-half of incorporated label was recovered in ethanol insoluble residues. Of this label, about 90% was solubilized by treatment, first with a preparation of pectinase-EDTA, then with dilute hydrochloric acid. The only labeled constituents in these hydrolyzates were d-galacturonic acid, d-glucuronic acid, 4-O-methyl-d-glucuronic acid, d-xylose, and l-arabinose, or larger oligosaccharide fragments containing these units. Medium external to excised root-tips grown under sterile conditions in myo-inositol-2-14C contained labeled polysaccharide. 相似文献
12.
13.
Characteristics of a Galactose-adapted Sugarcane Cell Line Grown in Suspension Culture 总被引:3,自引:3,他引:0
Although d-galactose is normally toxic to sugarcane (Saccharum sp.) cells, a cell line that grows on 100 mm galactose has been propagated. Nonadapted cells in a medium containing galactose instead of sucrose accumulate UDP-galactose; these cells also have much lower UDP-galactose 4-epimerase (EC 5.1.3.2) activity than do adapted cells. This enzyme may determine whether or not galactose will cause toxicity symptoms to develop. The growth rate of galactose-adapted cells is similar to most cell lines on several other carbohydrates. The galactose-adapted cells are also similar to sucrose stock cells in cell wall composition and sugar phosphate concentrations, but, like the nonadapted cells, accumulate free galactose. 相似文献
14.
Hidenori Imaki Toshifumi Tomoyasu Naoki Yamamoto Chiharu Taue Sachiko Masuda Ayuko Takao Nobuko Maeda Atsushi Tabata Robert A. Whiley Hideaki Nagamune 《Journal of bacteriology》2014,196(15):2817-2826
Streptococcus intermedius is a known human pathogen and belongs to the anginosus group (S. anginosus, S. intermedius, and S. constellatus) of streptococci (AGS). We found a large open reading frame (6,708 bp) in the lac operon, and bioinformatic analysis suggested that this gene encodes a novel glycosidase that can exhibit β-d-galactosidase and N-acetyl-β-d-hexosaminidase activities. We, therefore, named this protein “multisubstrate glycosidase A” (MsgA). To test whether MsgA has these glycosidase activities, the msgA gene was disrupted in S. intermedius. The msgA-deficient mutant no longer showed cell- and supernatant-associated β-d-galactosidase, β-d-fucosidase, N-acetyl-β-d-glucosaminidase, and N-acetyl-β-d-galactosaminidase activities, and all phenotypes were complemented in trans with a recombinant plasmid carrying msgA. Purified MsgA had all four of these glycosidase activities and exhibited the lowest Km with 4-methylumbelliferyl-linked N-acetyl-β-d-glucosaminide and the highest kcat with 4-methylumbelliferyl-linked β-d-galactopyranoside. In addition, the purified LacZ domain of MsgA had β-d-galactosidase and β-d-fucosidase activities, and the GH20 domain exhibited both N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities. The β-d-galactosidase and β-d-fucosidase activities of MsgA are thermolabile, and the optimal temperature of the reaction was 40°C, whereas almost all enzymatic activities disappeared at 49°C. The optimal temperatures for the N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities were 58 and 55°C, respectively. The requirement of sialidase treatment to remove sialic acid residues of the glycan branch end for glycan degradation by MsgA on human α1-antitrypsin indicates that MsgA has exoglycosidase activities. MsgA and sialidase might have an important function in the production and utilization of monosaccharides from oligosaccharides, such as glycans for survival in a normal habitat and for pathogenicity of S. intermedius. 相似文献
15.
A basic β-galactosidase (β-Galase) has been purified 281-fold from imbibed radish (Raphanus sativus L.) seeds by conventional purification procedures. The purified enzyme is an electrophoretically homogeneous protein consisting of a single polypeptide with an apparent molecular mass of 45 kilodaltons and pl values of 8.6 to 8.8. The enzyme was maximally active at pH 4.0 on p-nitrophenyl β-d-galactoside and β-1,3-linked galactobiose. The enzyme activity was inhibited strongly by Hg2+ and 4-chloromercuribenzoate. d-Galactono-(1→4)-lactone and d-galactal acted as potent competitive inhibitors. Using galactooligosaccharides differing in the types of linkage as the substrates, it was demonstrated that radish seed β-Galase specifically split off β-1,3- and β-1,6-linked d-galactosyl residues from the nonreducing ends, and their rates of hydrolysis increased with increasing chain lengths. Radish seed and leaf arabino-3,6-galactan-proteins were resistant to the β-galase alone but could be partially degraded by the enzyme after the treatment with a fungal α-l-arabinofuranosidase leaving some oligosaccharides consisting of d-galactose, uronic acid, l-arabinose, and other minor sugar components besides d-galactose as the main product. 相似文献
16.
Structural requirements for active intestinal transport. The nature of the carrier–sugar bonding at C-2 and the ring oxygen of the sugar
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Several weakly transported sugars were tested for transport by the Na+-dependent sugar carrier with slices of everted hamster intestinal tissue. Sugars were assumed to be transported by this carrier if the accumulation was diminished in the absence of Na+ and in the presence of the competitive inhibitor 1,5-anhydro-d-glucitol. The extent of accumulation was correlated with the number of hydroxyl groups in the d-gluco configuration if the ring oxygen was placed in the normal d-glucose position. 5-Thio-d-glucose, with a sulphur atom in the ring, was transported at about the same rate as d-glucose and had a similar Ki for d-galactose transport, but myoinositol was poorly accumulated. It is suggested that there is no hydrogen bonding at the ring oxygen atom, but that the oxygen atom is found at this position as a result of steric constraints. No sugar without a hydroxyl group in the d-gluco position at C-2 of the sugar, including d-mannose, 2-deoxy-d-glucose, 2-chloro-2-deoxy-d-glucose and 2-deoxy-2-fluoro-d-glucose, was transported by the Na+-dependent carrier, but these sugars and l-fucose weakly and competitively inhibit the Na+-dependent accumulation of l-glucose into slices of everted hamster intestinal tissue. It is concluded that the bond between the carrier and C-2 of the sugar may be covalent, and a possible mechanism for active intestinal transport is proposed. 相似文献
17.
Escherichia coli K-12 provided with glucose and a mixture of amino acids depletes l-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S l-serine deaminases (SdaA, SdaB, and TdcG) of E. coli K-12 is unable to do this. The high level of l-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C1 units and interferes with cell wall synthesis. We suggest that at high concentrations, l-serine decreases synthesis of UDP-N-acetylmuramate-l-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high l-serine is overcome in several ways: by a large concentration of l-alanine, by overproducing MurC together with a low concentration of l-alanine, and by overproducing FtsW, thus promoting septal assembly and also by overexpression of the glycine cleavage operon. S-Adenosylmethionine reduces lysis and allows an extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate and replicate their DNA, reaching at least 180 times their usual length, but cannot divide.The Escherichia coli genome contains three genes, sdaA, sdaB, and tdcG, specifying three very similar 4Fe4S l-serine deaminases. These enzymes are very specific for l-serine for which they have unusually high Km values (3, 32). Expression of the three genes is regulated so that at least one of the gene products is synthesized under all common growth conditions (25). This suggests an important physiological role for the enzymes. However, why E. coli needs to deaminate l-serine has been a long-standing problem of E. coli physiology, the more so since it cannot use l-serine as the sole carbon source.We showed recently that an E. coli strain devoid of all three l-serine deaminases (l-SDs) loses control over its size, shape, and cell division when faced with complex amino acid mixtures containing l-serine (32). We attributed this to starvation for single-carbon (C1) units and/or S-adenosylmethionine (SAM). C1 units are usually made from serine via serine hydroxymethyl transferase (GlyA) or via glycine cleavage (GCV). The l-SD-deficient triple mutant strain is starved for C1 in the presence of amino acids, because externally provided glycine inhibits GlyA and a very high internal l-serine concentration along with several other amino acids inhibits glycine cleavage. While the parent cell can defend itself by reducing the l-serine level by deamination, this crucial reaction is missing in the ΔsdaA ΔsdaB ΔtdcG triple mutant. We therefore consider these to be “defensive” serine deaminases.The fact that an inability to deaminate l-serine leads to a high concentration of l-serine and inhibition of GlyA is not surprising. However, it is not obvious why a high level of l-serine inhibits cell division and causes swelling, lysis, and filamentation. Serine toxicity due to inhibition of biosynthesis of isoleucine (11) and aromatic amino acids (21) has been reported but is not relevant here, since these amino acids are provided in Casamino Acids.We show here that at high internal concentrations, l-serine also causes problems with peptidoglycan synthesis, thus weakening the cell wall. Peptidoglycan is a polymer of long glycan chains made up of alternating N-acetylglucosamine and N-acetylmuramic acid residues, cross-linked by l-alanyl-γ-d-glutamyl-meso-diaminopimelyl-d-alanine tetrapeptides (1, 28). The glucosamine and muramate residues and the pentapeptide (from which the tetrapeptide is derived) are all synthesized in the cytoplasm and then are exported to be polymerized into extracellular peptidoglycan (2).In this paper, we show that lysis is caused by l-serine interfering with the first step of synthesis of the cross-linking peptide, the addition of l-alanine to uridine diphosphate-N-acetylmuramate. This interference is probably due to a competition between serine and l-alanine for the ligase, MurC, which adds the first l-alanine to UDP-N-acetylmuramate (7, 10, 15). As described here, the weakening of the cell wall by l-serine can be overcome by a variety of methods that reduce the endogenous l-serine pool or counteract the effects of high levels of l-serine. 相似文献
18.
Erin L. Westman David J. McNally Armen Charchoglyan Dyanne Brewer Robert A. Field Joseph S. Lam 《The Journal of biological chemistry》2009,284(18):11854-11862
The lipopolysaccharide of Pseudomonas aeruginosa PAO1 contains an
unusual sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid
(d-ManNAc3NAcA). wbpB, wbpE, and wbpD
are thought to encode oxidase, transaminase, and N-acetyltransferase
enzymes. To characterize their functions, recombinant proteins were
overexpressed and purified from heterologous hosts. Activities of
His6-WbpB and His6-WbpE were detected only when both
proteins were combined in the same reaction. Using a direct MALDI-TOF mass
spectrometry approach, we identified ions that corresponded to the predicted
products of WbpB (UDP-3-keto-d-GlcNAcA) and WbpE
(UDP-d-GlcNAc3NA) in the coupled enzyme-substrate reaction.
Additionally, in reactions involving WbpB, WbpE, and WbpD, an ion consistent
with the expected product of WbpD (UDP-d-GlcNAc3NAcA) was
identified. Preparative quantities of UDP-d-GlcNAc3NA and
UDP-d-GlcNAc3NAcA were enzymatically synthesized. These compounds
were purified by high-performance liquid chromatography, and their structures
were elucidated by NMR spectroscopy. This is the first report of the
functional characterization of these proteins, and the enzymatic synthesis of
UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA.Gram-negative organisms such as Pseudomonas aeruginosa produce
lipopolysaccharide
(LPS)4 as an essential
component of the outer leaflet of the outer membrane. LPS can be conceptually
divided into three parts: lipid A, which anchors LPS into the membrane; core
oligosaccharide, which contributes to membrane stability; and the O-antigen,
which is a polysaccharide that extends away from the cell surface. In P.
aeruginosa, two types of O-antigen are observed: A-band O-antigen, which
is common to most strains, and B-band O-antigen, which is variable and
therefore used as the basis of the International Antigenic Typing Scheme
(1). P. aeruginosa
serotypes O2, O5, O16, O18, and O20 collectively belong to serogroup O2,
because they all share common backbone sugar structures in their O-antigen
repeat units consisting of two di-N-acetylated uronic acids and one
2-acetamido-2,6-dideoxy-d-galactose
(N-acetyl-d-fucosamine). The minor structural variations
in the O-antigen repeat units that differentiate this serogroup into five
serotypes are: the type of glycosidic linkage between O-units (alpha
versus beta) that is formed by the O-antigen polymerase (Wzy),
isomers present (d-mannuronic or l-guluronic acid), and
acetyl group substituents
(2–4).
The B-band O-antigen of P. aeruginosa PAO1 (serotype O5) contains a
repeating trisaccharide of
2-acetamido-3-acetamidino-2,3-dideoxy-d-mannuronic acid
(d-ManNAc3NAmA),
2,3-diacetamido-2,3-dideoxy-d-mannuronic acid
(d-ManNAc3NAcA), and 2-acetamido-2,6-dideoxy-d-galactose
(3).The biosynthesis of the two mannuronic acid derivatives has yet to be fully
understood and has been the subject of investigation by our group. To produce
UDP-d-ManNAc3NAcA, a five-step pathway has been proposed
(Fig. 1) that requires the
products of five genes localized to the B-band O-antigen biosynthesis cluster
(5). The O-antigen biosynthesis
cluster was shown to be identical for all serotypes within serogroup O2, which
further underscores the high similarity between these serotypes
(5). The five genes, including
wbpA, wbpB, wbpE, wbpD, and wbpI, have been shown to be
essential for B-band LPS biosynthesis, because knockout mutants of each of
these genes are deficient in B-band O-antigen
(6–8).
Homologs of all five of the proteins required for the
UDP-d-ManNAc3NAcA biosynthesis pathway are conserved in other
bacterial pathogens, including Bordetella pertussis, Bordetella
parapertussis, and Bordetella bronchiseptica.
Cross-complementation of P. aeruginosa knockout mutants lacking
wbpA, wbpB, wbpE, wbpD, or wbpI with the homologues from
B. pertussis could fully restore LPS production in the P.
aeruginosa LPS mutants, suggesting that the genes from B.
pertussis are functional homologs of the wbp genes
(7). Homologs of these genes
could be identified in diverse bacterial species, demonstrating the importance
of UDP-d-ManNAc3NAcA biosynthesis beyond its role in P.
aeruginosa (7).Open in a separate windowFIGURE 1.Proposed pathway for the biosynthesis of UDP-d-ManNAc3NAcA in
P. aeruginosa PAO1. The full names of the sugars are as follows:
GlcNAc, 2-acetamido-2-deoxy-d-glucose; GlcNAcA,
2-acetamido-2-deoxy-d-glucuronic acid; 3-keto-d-GlcNAcA,
2-acetamido-2-deoxy-d-ribo-hex-3-uluronic acid; GlcNAc3NA,
2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid; GlcNAc3NAcA,
2,3-diacetamido-2,3-dideoxy-d-glucuronic acid; ManNAc3NAcA,
2,3-diacetamido-2,3-dideoxy-d-mannuronic acid. Adapted from Ref.
8.The first enzyme of the UDP-d-ManNAc3NAcA biosynthesis pathway,
WbpA, is a 6-dehydrogenase that converts
UDP-2-acetamido-2-deoxy-d-glucose
(N-acetyl-d-glucosamine; UDP-d-GlcNAc) to
UDP-2-acetamido-2-deoxy-d-glucuronic acid
(N-acetyl-d-glucosaminuronic acid,
UDP-d-GlcNAcA) using NAD+ as a coenzyme
(9)
(Fig. 1). Following this, the
second step in UDP-d-ManNAc3NAcA biosynthesis is proposed to be an
oxidation reaction catalyzed by WbpB, forming
UDP-2-acetamido-2-deoxy-d-ribo-hex-3-uluronic acid
(3-keto-d-GlcNAcA), which in turn is used as the substrate for
transamination by WbpE, creating
UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid
(d-GlcNAc3NA).This residue is thought to be the substrate for WbpD, a putative
N-acetyltransferase of the hexapeptide acyltransferase superfamily
(10) that requires acetyl-CoA
as a co-substrate (8). WbpD has
been proposed to synthesize
UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid
(UDP-d-GlcNAc-3NAcA), which is utilized in the B-band O-antigen of
P. aeruginosa serotype O1. In P. aeruginosa serogroup O2,
the UDP-d-GlcNAc3NAcA is then epimerized by WbpI to create the
UDP-d-ManNAc3NAcA required for incorporation into B-band LPS
(11). A derivative of
UDP-d-ManNAc3NAcA is also used in the synthesis of B-band O-antigen
of P. aeruginosa serogroup O2. UDP-d-ManNAc3NAmA is
thought to be produced through additional modification of
UDP-d-ManNAc3NAcA via the action of WbpG, an amidotransferase,
which has also been demonstrated to be essential for the production of B-band
O-antigen (12,
13).In the current study, our aim was to define the function of WbpB, WbpE, and
WbpD, because only genetic evidence has previously been given for the
involvement of wbpB and wbpE
(7), and the reaction catalyzed
by WbpD could not be demonstrated due to the unavailability of its presumed
substrate, UDP-d-GlcNAc3NA
(8). The functional
characterization of these proteins is also important for understanding LPS
biosynthesis in B. pertussis, because the genes in the LPS locus of
this species, wlbA, wlbC, and wlbB, could cross-complement
knockouts of wbpB, wbpE, and wbpD, respectively, when
expressed in P. aeruginosa PAO1
(7). Furthermore, these three
proteins form a cassette for the generation of C-3 N-acetylated
hexoses and may be important for the biosynthesis of a variety of other
sugars. Capillary electrophoresis and MALDI-TOF mass spectrometry were used to
analyze reaction mixtures of WbpB and WbpE and showed that the expected
products were produced only when both enzymes were present together. Achieving
the enzymatic synthesis of the product of both enzymes, which was demonstrated
to be UDP-d-GlcNAc3NA by 1H NMR spectroscopy, was a key
breakthrough, because this rare sugar has never before been produced by any
means. UDP-d-GlcNAc3NA was also essential for use as the substrate
of WbpD, which not only allowed us to determine the enzymatic activity of this
protein but also allowed the enzymatic synthesis of
UDP-d-GlcNAc3NAcA to be achieved as well. Although this sugar had
previously been produced through a 17-step chemical synthesis
(11,
14), the 4-step concurrent
enzymatic reaction demonstrates the advantage of linking chemistry with
biology and represents a significant saving of both time and reagents as
compared with chemical synthesis. Finally, our data also showed the success in
reconstituting in vitro the 5-step pathway for the biosynthesis of
UDP-d-ManNAc3NAcA in P. aeruginosa. 相似文献
19.
Dietrich O. R. Keppler Christa Schulz-Holstege Joachim Fauler Karl A. Reiffen Friedhelm Schneider 《The Biochemical journal》1982,206(1):139-146
d-Galactosone (d-lyxo-2-hexosulose) is phosphorylated and metabolized to the uridine diphosphate derivative in AS-30D hepatoma cells and rat liver. These reactions were catalysed in vitro by galactokinase and hexose-1-phosphate uridylyltransferase. Nucleotide analyses by high-performance liquid chromatography and enzymic assays revealed that this galactose analogue interferes with cellular pyrimidine nucleotide metabolism leading to a deficiency of UTP. [14C]Uridine labelling of hepatoma cells indicated a division of [14C]uridylate from UTP into UDP-galactosone; the latter was formed at a rate of more than 1.7mmol×h−1×(kg AS-30D or liver wet wt.)−1. As a consequence of UTP deficiency, d-galactosone (1mmol/1 or 1mmol/kg body wt.) strongly enhanced the rate of pyrimidine synthesis de novo as evidenced by incorporation of 14CO2 into uridylate and by an expansion of the uridylate pool. This resulted in a doubling of the total acid-soluble uridylate pool within 70min in the hepatoma cells and within 110min in rat liver. Combined treatment of hepatoma cells with d-galactosone and N-(phosphonoacetyl)-l-aspartate, an inhibitor of aspartate carbamoyltransferase, prevented the expansion of the uridylate pool and led to a synergistic reduction of UTP to 10% of the content in control cells. Hepatic UTP deficiency was selective with respect to other nucleotide 5′-triphosphates but was associated with reduced contents of UDP-glucose, UDP-glucuronate, and UDP-N-acetylhexosamines. Isolation of the UDP derivative of d-galactosone revealed an extremely alkali-labile UDP-sugar, probably an isomerization product of UDP-galactosone, that was degraded by elimination of UDP with a half-life of 45min at pH7.5 and 37°C. The instability of UDP-galactosone may contribute in vivo to limit the time period of severe uridine phosphate deficiency in addition to the compensatory role of pyrimidine synthesis de novo. During the initial time period, however, d-galactosone is effective as a powerful uridylate-trapping sugar analogue. 相似文献
20.
The transport of some sugars at the antiluminal face of renal cells was studied using teased tubules of flounder (Pseudopleuronectes americanus). The analytical procedure allowed the determination of both free and total (free plus phosphorylated) tissue sugars. The inulin space of the preparation was 0.333 ± 0.017 kg/kg wet wt (7 animals, 33 analyses). The nonmetabolizable α-methyl-D-glucoside entered the cells by a carrier-mediated (phloridzin-sensitive), ouabain-insensitive process. The steady-state tissue/medium ratio was systematically below that for diffusion equilibrium. D-Glucose was a poor inhibitor of α-methyl-glucoside transport, D-galactose was ineffective. The phloridzin-sensitive transport processes of 2-deoxy-D-glucose,D-galactose,and 2-deoxy-D-galactose were associated with considerable phosphorylation. Kinetic evidence suggested that these sugars were transported in free form and subsequently were phosphorylated. 2-Deoxy-D-glucose accumulated in the cells against a slight concentration gradient. This transport was greatly inhibited by D-glucose, whereas α-methyl-glucoside and also D-galactose and its 2-deoxy-derivative were ineffective. D-Galactose and 2-deoxy-D-galactose mutually competed for transport; D-glucose, 2-deoxy-D-glucose, and α-methyl-D-glucoside were ineffective. Studies using various sugars as inhibitors suggest the presence of three carrier-mediated pathways of sugar transport at the antiluminal cell face of the flounder renal tubule: the pathway of α-methyl-D-glucoside (not shared by D-glucose); the pathway commonly shared by 2-deoxy-D-glucose and D-glucose; the pathway shared by D-galactose and 2-deoxy-D-galactose. 相似文献