Prediction of three-dimensional structures of enzyme-substrate and enzyme-inhibitor complexes of lysozyme. (57/1610)

Conformational energy calculations were used to predict the three-dimensional structures of enzyme-substrate and enzyme-inhibitor complexes of lysozyme. A global search method, involving the use of a disaccharide fragment molecule, was used initially to determine all favorable binding regions at the active site. It is shown that the binding of a series of (nonfragmented) oligomers of N-acetylglucosamine is highly specific. The results show further that (a) the enzyme recognizes only one backbone conformation of the oligomer, corresponding to a left-handed helix, and (b) for saccharides containing two or more N-acetylglucosamine residues, two residues bind preferentially to the C and D sites. The calculations also suggest that the chair form of N-acetylglucosamine can bind to the D region. The saccharide residues of tetra-N-acetylglucosamine bind to the A-B-C-D sites, with the residues at the A-B-C sites having essentially the same conformation and orientation as those in the x-ray structure of tetra-N-acetylglucosamine-delta-lactone bound to lysozyme.  (+info)

Biosynthesis of peptidoglycan in Pseudomonas aeruginosa. 2. Mode of action of beta-lactam antibiotics. (58/1610)

The intrinsic effect of various beta-lactam antibiotics on the biosynthesis of peptidoglycan of Pseudomonas aeruginosa X-48 was investigated. Most of the cephalosporins and penicillins tested already at 0.5 microgram/ml strongly inhibited (a) the incorporation of nascent peptidoglycan into the detergent-insoluble fraction (greater than 75%), (b) the formation of peptide crosslinkages (greater than 60%) and (c) the activity of the DD-carboxypeptidase and partially that of the transpeptidase (approximately 90% and approximately 40% respectively). Another group of beta-lactum drugs did not inhibit incorporation into the material insoluble in sodium dodecylsulfate, the formation of peptide crosslinkages nor transpeptidase activity. They only partially inhibited the activity of the DD-carboxypeptidase--endopeptidase system (40--50% at 0.5 microgram/ml). The results obtained differ from those of Presslitz and Ray [Antimicrob, Agents Chemother. 7, 578--581 (1975)] and show some resemblance to the effects of beta-lactams on the biosynthesis of Escherichia coli peptidoglycan.  (+info)

Leguminous lectins as tools for studying the role of sugar residues in leukocyte recruitment. (59/1610)

The natural physiological ligands for selectins are oligosaccharides found in glycoprotein or glycolipid molecules in cell membranes. In order to study the role of sugar residues in the in vivo lectin anti-inflammatory effect, we tested three leguminous lectins with different carbohydrate binding affinities in the peritonitis and paw oedema models induced by carrageenin in rats. L. sericeus lectin was more anti-inflammatory than D. virgata lectin, the effects being reversed by their specific binding sugars (N-acetylglucosamine and alpha-methylmannoside, respectively). However, V. macrocarpa, a galactose-specific lectin, was not anti-inflammatory. The proposed anti-inflammatory activity of lectins could be due to a blockage of neutrophil-selectin carbohydrate ligands. Thus, according to the present data, we suggest an important role for N-acetylglucosamine residue as the major ligand for selectins on rat neutrophil membranes.  (+info)

Glucose stimulates protein modification by O-linked GlcNAc in pancreatic beta cells: linkage of O-linked GlcNAc to beta cell death. (60/1610)

The pancreatic beta cell can respond in the long term to hyperglycemia both with an increased capacity for insulin production and, in susceptible individuals, with apoptosis. When glucose-induced apoptosis offsets the increasing beta cell capacity, type 2 diabetes results. Here, we tested the idea that the pathway of glucose metabolism that leads to the modification of intracellular proteins with the O-linked monosaccharide N-acetylglucosamine (O-GlcNAc) is involved in the glucose-induced apoptosis. This idea is based on two recent observations. First, the beta cell expresses much more O-GlcNAc transferase than any other known cell, and second, that the beta cell-specific toxin, streptozotocin (STZ), itself a GlcNAc analog, specifically blocks the enzyme that cleaves O-GlcNAc from intracellular proteins. As a consequence, we now show that hyperglycemia leads to the rapid and reversible accumulation of O-GlcNAc specifically in beta cells in vivo. Animals pretreated with STZ also accumulate O-GlcNAc in their beta cells when hyperglycemic, but this change is sustained upon re-establishment of euglycemia. In concert with the idea that STZ toxicity results from the sustained accumulation of O-GlcNAc after a hyperglycemic episode, we established a low-dose STZ protocol in which the beta cells' toxicity of STZ was manifest only after glucose or glucosamine administration. Transgenic mice with impaired beta cell glucosamine synthesis treated with this protocol are resistant to the diabetogenic effect of STZ plus glucose yet succumb to STZ plus glucosamine. This study provides a causal link between apoptosis in beta cells and glucose metabolism through glucosamine to O-GlcNAc, implicating this pathway of glucose metabolism with beta cell glucose toxicity.  (+info)

Cyborg lectins: novel leguminous lectins with unique specificities. (61/1610)

Bauhinia purpurea lectin (BPA) is one of the beta-galactose-binding leguminous lectins. Leguminous lectins contain a long metal-binding loop, part of which determines their carbohydrate-binding specificities. Random mutations were introduced into a portion of the cDNA coding BPA that corresponds to the carbohydrate-binding loop of the lectin. An library of the mutant lectin expressed on the surface of lambda foo phages was screened by the panning method. Several phage clones with an affinity for mannose or N-acetylglucosamine were isolated. These results indicate the possibility of making artificial lectins (so-called "cyborg lectins") with distinct and desired carbohydrate-binding specificities.  (+info)

Characterization of Staphylococcus aureus cell wall glycan strands, evidence for a new beta-N-acetylglucosaminidase activity. (62/1610)

Using sequential digestion with the glycyl-glycine endopeptidase lysostaphin followed by the pneumococcal N-acetylmuramyl-L-alanine amidase (amidase), the glycan strands of the peptidoglycan of Staphylococcus aureus were purified and analyzed by a combination of reverse-phase-high pressure liquid chromatography (HPLC) and mass spectrometry. Reverse-phase-HPLC resolved the glycan strands to a family of major peaks, which represented oligosaccharides composed of repeating disaccharide units (N-acetylglucosamine-[beta-1, 4]-N-acetylmuramic acid) with different degrees of polymerization and terminating with N-acetylmuramic acid residues at the reducing ends. The method allowed separation of strands up to 23-26 disaccharide units with a predominant length between 3 and 10 and an average degree of polymerization of approximately 6. Glycan strands with a higher degree of polymerization (>26 disaccharide units) represented 10-15% of the total UV absorbing glycan material. A unique feature of the staphylococcal glycan strands was the presence of minor satellite peaks that were present throughout the HPLC elution profile eluting either just prior or shortly after the major oligosaccharide peaks. A number of observations including mass spectrometric analysis suggest that the satellites are the products of an N-acetylglucosaminidase activity that differs from the atl gene product and that appears to be involved with modification of the glycan strand structure.  (+info)

The X-ray structure of a chitinase from the pathogenic fungus Coccidioides immitis. (63/1610)

The X-ray structure of chitinase from the fungal pathogen Coccidioides immitis has been solved to 2.2 A resolution. Like other members of the class 18 hydrolase family, this 427 residue protein is an eight-stranded beta/alpha-barrel. Although lacking an N-terminal chitin anchoring domain, the enzyme closely resembles the chitinase from Serratia marcescens. Among the conserved features are three cis peptide bonds, all involving conserved active site residues. The active site is formed from conserved residues such as tryptophans 47, 131, 315, 378, tyrosines 239 and 293, and arginines 52 and 295. Glu171 is the catalytic acid in the hydrolytic mechanism; it was mutated to a Gln, and activity was abolished. Allosamidin is a substrate analog that strongly inhibits the class 18 enzymes. Its binding to the chitinase hevamine has been observed, and we used conserved structural features of the two enzymes to predict the inhibitors binding to the fungal enzyme.  (+info)

Differentiation of O-acetyl and O-carbamoyl esters of N-acetyl-glucosamine by decomposition of their oxonium ions. Application to the structure of the nonreducing terminal residue of Nod factors. (64/1610)

Nod factors are substituted N-acyl chito-oligomers secreted by plant symbiotic bacteria of the Rhizobium family. Substitutions on the oligosaccharide core specify their recognition by host plants. A method using tandem mass spectrometry is proposed to locate the O-acetyl and O-carbamoyl substituents on the nonreducing terminal residue of the chito-oligomers. As model compounds, all the positional isomers of monoacetyl and monocarbamoyl esters of 1-O-methyl-N-acetyl-alpha-D-glucosamine were synthesized. Oxonium ions (MH - CH3OH)+ were generated by liquid secondary ion mass spectrometry (LSIMS) and their decomposition was recorded on a tandem magnetic instrument. Large differences were observed in the relative abundances of ions resulting from elimination of water and of the O-ester substituent from metastable oxonium ions. Deuterium exchange reactions indicated parallel elimination pathways involving either exchangeable or carbon-linked hydrogens. The intensity ratios of some of the ions generated by collisions with helium atoms allowed the isomers to be distinguished. The main dissociation routes were identified. Metastable and collision-induced decomposition of the B1 ions from Nod factors of Sinorhizobium meliloti and Azorhizobium caulinodans resembled that of the 6-O-substituted N-acetylglucosamine models. Decomposition of the B1 ion from Mesorhizobium loti and Rhizobium etli Nod factors, was similar to that of 3-O-carbamoyl N-acetyl-glucosamine and different to that of the 4-O isomer. 6-O- and 3-O-carbamoylation specified by the nodU and nolO genes, respectively, of Rhizobium. sp. NGR234 were confirmed.  (+info)