bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural heterogeneity in the O polysaccharide of Pseudomonas syringae pv. coriandricola GSPB 2028 (NCPPB 3780, W‐43)
Authors: Knirel Yuriy A. , Ovod Vladimir V. , Paramonov Nikolay A. , Krohn Kai J. 
Published: March 2003

The O polysaccharide (OPS) of the lipopolysaccharide of Pseudomonas syringae pv. coriandricola GSPB 2028 (NCPPB 3780, W‐43) was studied by Smith degradation and 1H‐NMR and 13C‐NMR spectroscopy, including two‐dimensional COSY, TOCSY, NOESY, and H‐detected 1H,13C heteronuclear multiple‐quantum coherence (HMQC) experiments. The OPS was shown to consist of pentasaccharide O repeats of two types both containing four
L‐rhamnose and one 3‐acetamido‐3,6‐dideoxy‐
D‐galactose (
D‐Fuc3NAc) residue. Structure 1 of the major O repeat which had been established earlier [Das, S., Ramm, M., Kochanowski, H. & Basu, S. (1994) J. Bacteriol. 176, 6550−6557], was confirmed by our data, and a new structure 2 was elucidated for the minor O repeat and found to differ from the structure 1 only in the position of substitution of one of the rhamnose residues in the main chain.α‐ D‐Fucp3NAc1↓2←2)‐α‐ L‐Rhap‐(1←2)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1← 1 α‐ D‐Fucp3NAc1↓2←3)‐α‐ L‐Rhap‐(1←2)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1← 2 A role of structural and immunochemical features of the LPS for defining the taxonomical position of the bacterium studied is discussed.
Desulfovibrio gigas neelaredoxin
Authors: Silva Gabriela , Oliveira Solange , Gomes Cláudio M. , Pacheco Isabel , Liu Ming Y. , Xavier António V. , Teixeira Miguel , LeGall Jean , Rodrigues‐Pousada Claudina 
Published: March 2003

Neelaredoxin, a small non‐heme blue iron protein from the sulfate‐reducing bacterium Desulfovibrio gigas[Chen, L., Sharma, P., LeGall, J., Mariano, A.M., Teixeira M. and Xavier, A.V. (1994) Eur. J. Biochem.226, 613–618] is shown to be encoded by a polycistronic unit which contains two additional open reading frames (ORF‐1 and ORF‐2) coding for chemotaxis‐like proteins. ORF‐1 has domains highly homologous with those structurally and functionally important in methyl‐accepting chemotaxis proteins, including two putative transmembrane helices, potential methylation sites and the interaction domain with CheW proteins. Interestingly, ORF‐2 encodes a protein having homologies with CheW proteins. Neelaredoxin is also shown to have significant superoxide dismutase activity (1200 U·mg–1), making it a novel type of iron superoxide dismutase. Analysis of genomic data shows that neelaredoxin‐like putative polypeptides are present in strict anaerobic archaea, suggesting that this is a primordial superoxide dismutase. The three proteins encoded in this operon may be involved in the oxygen‐sensing mechanisms of this anaerobic bacterium, indicating a possible transcriptional mechanism to sense and respond to potential stress agents.
Structural elucidation of a novel exopolysaccharide produced by a mucoid clinical isolate of Burkholderia cepacia
Authors: Cérantola Stéphane , Lemassu‐Jacquier Anne , Montrozier Henri 
Published: March 2003

The structure of the exopolysaccharide (EPS) produced by a clinical isolate of Burkholderia cepacia isolated from a patient with fibrocystic lung disease has been investigated. By means of methylation analyses, carboxyl reduction, partial depolymerization by fuming HCl and chemical degradations such as Smith degradation, lithium‐ethylenediamine degradation and β‐elimination, supported by GC/MS and NMR spectroscopic analyses, the repeat unit of the EPS has been identified and was shown to correspond to the acidic branched heptasaccharide with the following structure:This partially acetylated acidic polymer, distinguished by the presence of the less usual d‐isomer of rhamnose and of a trisubstituted glucuronic acid residue, could represent the main EPS produced by this bacterial species.
The structures of the carbohydrate backbones of the lipopolysaccharides from Escherichia coli rough mutants F470 (R1 core type) and F576 (R2 core type)
Authors: Vinogradov Evgeny V. , van der Drift Koen , Thomas‐Oates Jane E. , Meshkov Sergey , Brade Helmut , Holst Otto 
Published: March 2003

The lipopolysaccharides (LPS) from Escherichia coli rough mutant strains F470 (R1 core type) and F576 (R2 core type) were deacylated yielding in each case a mixture of oligosaccharides with one predominant product which was isolated using high‐performance anion‐exchange chromatography. In addition, one oligosaccharide present in minor quantities was isolated from LPS of E. coli strain F576 (R2 core type). The structures of the oligosaccharides were determined by chemical analyses and NMR spectroscopic experiments. Furthermore, de‐O‐acylated and dephosphorylated LPS preparations were investigated by fast‐atom bombardment and collision induced dissociation tandem mass spectrometry. The combined data allow us to deduce the following carbohydrate backbones of the E. coli R1 and R2 core types which share the following structure (Scheme 1):but differ in the substituents R1 and R2 which for the R1 core type are predominantly:and to a minor extent:and for the R2 core type predominantly:and to a minor extent:in which all sugars are d‐pyranoses (l,d‐Hep, lglycerodmanno‐heptopyranose; P, phosphate).
Acetylene hydratase of Pelobacter acetylenicus
Authors: Meckenstock Rainer U. , Krieger Robert , Ensign Scott , Kroneck Peter M. H. , Schink Bernhard 
Published: March 2003

Acetylene hydratase of Pelobacter acetylenicus is a tungsten iron–sulfur protein involved in the fermentation of acetylene to ethanol and acetate. Expression of the enzyme was increased 10‐fold by feeding a 50‐L batch culture continuously with 104 Pa acetylene at pH 6.8–7.0. Acetylene hydratase was purified to homogeneity by a three‐step procedure in either the absence or presence of dioxygen. The enzyme was a monomer with a molecular mass of 73 kDa (SDS/PAGE) or 83 kDa (matrix‐assisted laser‐desorption ionization MS) and contained 0.5 ± 0.1 W (inductively coupled plasma/MS) and 1.3 ± 0.1 molybdopterin–guanine dinucleotide per mol. Selenium was absent. EPR spectra (enzyme as isolated, under air) showed a signal typical of a [3Fe–4S] cluster with gav = 2.01, at 10 K. In enzyme prepared under N2/H2, this signal was absent and reaction with dithionite led to a rhombic signal with gz = 2.048, gy = 1.939 and gx = 1.920 indicative of a low‐potential ferredoxin‐type [4Fe–4S] cluster. Upon oxidation with hexacyanoferrate(III), a new signal appeared with gx = 2.007, gy = 2.019 and gz = 2.048 (gav = 2.022), which disappeared after further oxidation. The signal was still visible at 150 K and was tentatively assigned to a W(V) center. The iron–sulfur center of acetylene hydratase (prepared under N2/H2) gave a midpoint redox potential of −410 ± 20 mV in a spectrophotometric titration with dithionite. Enzyme activity depended on the redox potential of the solution, with 50% of maximum activity at −340 ± 20 mV. The presence of a pterin–guanine dinucleotide cofactor differentiates acetylene hydratase from the aldehyde ferredoxin oxidoreductase‐type enzymes which have a pterin mononucleotide cofactor.
Placenta‐specific transcripts of the aromatase encoding gene include different untranslated first exons in sheep and cattle
Authors: Vanselow Jens , Zsolnai Attila , Fésüs Laszlo , Fürbass Rainer , Schwerin Manfred 
Published: March 2003

The aim of the present study was the characterization of the ovine aromatase cytochrome P450 encoding gene (Cyp19) and the analysis of its tissue‐specific expression. Two loci with considerable sequence identity were found (Cyp19 and Cyp19b). From Cyp19, tissue‐specific transcript variants with different untranslated first exons but identical coding regions could be identified. Cyp19b transcripts were not detected. In the sheep brain and ovarian granulosa cells transcript variants, starting with the untranslated exons 1.4 and 2, respectively, were preferentially found. Exons 1.2 and 1.3 which had been described in bovines could not be detected in sheep and the major 5′ untranslated region of the bovine placental transcript, exon 1.1, was also not found to predominate in the sheep placenta. However this exon frequently was combined with a new untranslated exon (exon 1.1a) thus generating an alternative splice variant. The main placental transcripts in sheep had a different first exon (exon 1.5). Two alternatively spliced variants of this transcript were found with tissue‐specific preference. From the present data it can be concluded: (a) that the ovine genome contains two copies of Cyp19 of which only one is transcribed and may encode a functional protein; and (b) that in spite of being closely related species, sheep and cattle have remarkable differences concerning tissue‐specific transcript distribution and presumable promoter usage.
The effect of pressure and guanidine hydrochloride on azurins mutated in the hydrophobic core
Authors: Mei Giampiero , Di Venere Almerinda , Campeggi Flaminia Malvezzi , Gilardi Gianfranco , Rosato Nicola , De Matteis Fabio , Finazzi‐Agrò Alessandro 
Published: March 2003

The unfolding of the blue‐copper protein azurin from Pseudomonas aeruginosa by guanidine hydrochloride, under nonreducing conditions, has been studied by fluorescence techniques and circular dichroism. The denaturation transition may be fitted by a simple two‐state model. The total free energy change from the native to the unfolded state was 9.4 ± 0.4 kcal·mol−1, while a lower value (6.4 ± 0.4 kcal·mol−1) was obtained for the metal depleted enzyme (apo‐azurin) suggesting that the copper atom plays an important stabilization role. Azurin and apo‐azurin were practically unaffected by hydrostatic pressure up to 3000 bar. Site‐directed mutagenesis has been used to destabilize the hydrophobic core of azurin. In particular either hydrophobic residue Ile7 or Phe110 has been substituted with a serine. The free energy change of unfolding by guanidinium hydrochloride, resulted to be 5.8 ± 0.3 kcal·mol−1 and 4.8 ± 0.3 kcal·mol−1 for Ile7Ser and Phe110Ser, respectively, showing that both mutants are much less stable than the wild‐type protein. The mutated apoproteins could be reversible denatured even by high pressure, as demonstrated by steady‐state fluorescence measurements. The change in volume associated to the pressure‐induced unfolding was estimated to be −24 mL·mol−1 for Ile7Ser and −55 mL·mol−1 for Phe110Ser. These results show that the tight packing of the hydrophobic residues that characterize the inner structure of azurin is fundamental for the protein stability. This suggests that the proper assembly of the hydrophobic core is one of the earliest and most crucial event in the folding process, bearing important implication for de novo design of proteins.
Differential characteristics of human 15‐lipoxygenase isozymes and a novel splice variant of 15S‐lipoxygenase
Authors: Kilty Iain , Logan Alison , Vickers Philip J. 
Published: March 2003

The lipoxygenases (LOs) are a family of nonheme iron dioxygenases that catalyse the insertion of molecular oxygen into polyunsaturated fatty acids. Five members of this gene family have been described in man, 5‐LO, 12S‐LO, 12R‐LO, 15‐LO and 15S‐LO. Using partially purified recombinant 15S‐LO enzyme and cells constitutively expressing this protein, we have compared the activity, substrate specificity, kinetic characteristics and regulation of this enzyme to that previously reported for 15‐LO. 15S‐LO has a threefold higher Km, similar Vmax and increased specificity of oxygenation for arachidonic acid, and a similar Km but decreased Vmax for linoleic acid in comparison to 15‐LO. Unlike 15‐LO, 15S‐LO is not suicide inactivated by the products of fatty acid oxygenation. However, in common with other LOs, 15S‐LO activity is regulated through calcium‐dependent association of the enzyme with the membrane fraction of cells.In addition, whilst independently cloning the recently described 15S‐LO, we identified a splice variant containing an in‐frame 87‐bp deletion corresponding to amino acids 401–429 inclusive. Modelling of the 15S‐LO and subsequent studies with partially purified recombinant protein suggest that the deleted region comprises a complete α‐helix flanking the active site of the enzyme resulting in decreased specificity of oxygenation and affinity for fatty acid substrates.Alternative splicing of 15S‐LO would therefore provide a further level of regulation of fatty acid metabolism. These results demonstrate that there are substantial differences in the enzyme characteristics and regulation of the 15‐LO isozymes which may reflect differing roles for the proteins in vivo.
NMR solution structure of a novel thioredoxin from Bacillus acidocaldarius
Authors: Nicastro Giuseppe , de Chiara Cesira , Pedone Emilia , Tatò Marco , Rossi Mosè , Bartolucci Simonetta 
Published: March 2003

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx), an eubacterium growing optimally at 333 K, is the first Trx described to date from a moderate thermophilic source. To understand the molecular basis of its thermostability, the three‐dimensional structure in the oxidized form was determined by NMR methods. A total of 2276 1H‐NMR derived distance constraints along with 23 hydrogen‐bonds, 72 φ and 27 χ1 torsion angle restraints, were used in a protocol employing simulated annealing followed by restrained molecular dynamics and restrained energy minimization.BacTrx consists of a well‐defined core region of five strands of β‐sheet, surrounded by four exposed α‐helices, features shared by other members of the thioredoxin family. The BacTrx 3D structure was compared with the Escherichia coli Trx (EcTrx) determined by X‐ray crystallographic diffraction, and a number of structural differences were observed that may contribute to its thermostabilty. The results of structural analysis indicated that protein stability is due to cumulative effects, the main factor being an increased number of ionic interactions cross‐linking different secondary structural elements and clamping the C‐terminal α‐helix to the core of the protein.
The heme‐independent manganese‐peroxidase activity depends on the presence of the C‐terminal domain within the Streptomyces reticuli catalase‐peroxidase CpeB
Authors: Zou Peijian , Schrempf Hildgund 
Published: March 2003

Streptomyces reticuli produces a heme‐containing homodimeric enzyme (160 kDa), the catalase‐peroxidase CpeB, which is processed to the enzyme CpeC during prolonged growth. CpeC contains four subunits of 60 kDa each that do not include the C‐terminal portion of the progenitor subunits. A genetically engineered cpeB gene encodes a truncated subunit lacking 195 of the C‐terminal amino acids; four of these subunits assemble to form the enzyme CpeD. Heme binds most strongly in CpeB, least in CpeD. The catalase‐peroxidase CpeB and its apo‐form (obtained after extraction of heme) catalyze the peroxidation of Mn(II) to Mn(III), independent of the presence or absence of the heme inhibitor KCN. CpeC and CpeD, in contrast, do not exhibit manganese‐peroxidase activity.The data show for the first time that a bacterial catalase‐peroxidase has a heme‐independent manganese‐peroxidase activity, which depends on the presence of the C‐terminal domain.
Recursive use of evolutionary conservation data in molecular modeling of membrane proteins
Authors: Torres Jaume , Arkin Isaiah T. 
Published: March 2003

Membrane proteins are currently the most biomedically important family of proteins, serving as targets for the majority of pharmaceutical agents. It is also clear that they are invariably abundant in all of the genomes sequence so far, representing up to a third of all open reading frames. Finally, and regrettably, it is clear that they are highly resistant to structural elucidation, representing less than 0.2% of the Protein Data Bank. Recent accomplishments in genome sequencing efforts, however, may help offset this imbalance through the availability of evolutionary conservation data.Herein, we develop a novel approach, utilizing a combination of evolutionary conservation data and global searching molecular dynamics simulations to model membrane proteins, deriving a model for the multidrug H+ antiporter EmrE, a transmembrane four‐helix bundle. Structures resulting from an extensive, rotational molecular dynamics search, were evaluated by comparing the residue specific interaction energy and the evolutionary conservation data. Subsequent rounds of molecular dynamics, in which confinement of the search space was undertaken in order to achieve a self consistent result, point to a structure that best satisfies the evolutionary conservation data. As the conservation patterns calculated for each of the helices suggested that the different conservation pattern for helix 3 (as well as being the most conserved) might be due to the oligomeric nature of EmrE, a dodecamer of helices was constructed based on the result of a search of helix 3 as a trimer. The resulting interaction energy per residue in the final model is in reasonable agreement with the evolutionary data and consistent with recent site directed mutagenesis experiments, pointing to the strength of this method as a general tool.
In vitro inhibition and intracellular enhancement of lysosomal α‐galactosidase A activity in Fabry lymphoblasts by 1‐deoxygalactonojirimycin and its derivatives
Authors: Asano Naoki , Ishii Satoshi , Kizu Haruhisa , Ikeda Kyoko , Yasuda Kayo , Kato Atsushi , Martin Olivier R. , Fan Jian‐Qiang 
Published: March 2003

Fabry disease is a lysosomal storage disorder caused by deficient lysosomal α‐galactosidase A (α‐Gal A) activity. Deficiency of the enzyme activity results in progressive deposition of neutral glycosphingolipids with terminal α‐galactosyl residue in vascular endothelial cells. We recently proposed a chemical chaperone therapy for this disease by administration of 1‐deoxygalactonojirimycin, a potent inhibitor of the enzyme, at subinhibitory intracellular concentrations [Fan, J.‐Q., Ishii, S., Asano, N. and Suzuki, Y. (1999) Nat. Med.5, 112–115]. 1‐Deoxygalactonojirimycin served as a specific chaperone for those mutant enzymes that failed to maintain their proper conformation to avoid excessive degradation. In order to establish a correlation between in vitro inhibitory activity and intracellular enhancement activity of the specific chemical chaperone, a series of 1‐deoxygalactonojirimycin derivatives were tested for activity with both α‐Gal A and Fabry lymphoblasts. 1‐Deoxygalactonojirimycin was the most potent inhibitor of α‐Gal A with an IC50 value of 0.04 µm. α‐Galacto‐homonojirimycin, α‐allo‐homonojirimycin and β‐1‐C‐butyl‐deoxygalactonojirimycin were effective inhibitors with IC50 values of 0.21, 4.3 and 16 µm, respectively. N‐Alkylation, deoxygenation at C‐2 and epimerization at C‐3 of 1‐deoxygalactonojirimycin markedly lowered or abolished its inhibition toward α‐Gal A. Inclusion of 1‐deoxygalactonojirimycin, α‐galacto‐homonojirimycin, α‐allo‐homonojirimycin and β‐1‐C‐butyl‐deoxygalactonojirimycin at 100 µm in culture medium of Fabry lymphoblasts increased the intracellular α‐Gal A activity by 14‐fold, 5.2‐fold, 2.4‐fold and 2.3‐fold, respectively. Weaker inhibitors showed only a minimum enhancement effect. These results suggest that more potent inhibitors act as more effective specific chemical chaperones for the mutant enzyme, and the potent competitive inhibitors of α‐Gal A are effective specific chemical chaperones for Fabry disease.
Distribution, cloning, and characterization of porcine nucleoside triphosphate diphosphohydrolase‐1
Authors: Lemmens Raf , Vanduffel Luc , Kittel Agnes , Beaudoin Adrien R. , Benrezzak Ouhida , Sévigny Jean 
Published: March 2003

In this study, we have investigated the distribution of the enzyme nucleoside triphosphate diphosphohydrolase‐1 (NTPDase1; EC 3.6.1.5) in a subset of pig tissues by biochemical activity and Western blotting with antibodies against porcine NTPDase1. The highest expression of this enzyme was found in vascular endothelium, smooth muscle, spleen and lung.The complete cDNA of NTPDase1 from aorta endothelial cells was sequenced using primer walking. The protein consists of 510 amino acids, with a calculated molecular mass of 57 756 Da. The amino‐acid sequence indicated seven putative N‐glycosylation sites and one potential intracellular cGMP‐ and cAMP‐dependent protein kinase phosphorylation site. As expected, the protein has a very high homology to other known mammalian ATPDases and CD39 molecules, and includes all five apyrase conserved regions.Expression of the complete cDNA in COS‐7 cells confirmed that NTPDase1 codes for a transmembrane glycoprotein with ecto‐ATPase and ecto‐ADPase activities. Two proteolytic products of NTPDase1, with molecular mass of 54 and 27 kDa, respectively, were consistently present in proteins from transfected COS‐7 cells and in particulate fractions from different tissues. A trypsin cleavage site, giving rise to these two cleavage products, was identified. In order to remain enzymatically active, the two cleavage products have to interact by non–covalent interactions.
Structural investigations of the hemoglobin of the cyanobacterium Synechocystis PCC6803 reveal a unique distal heme pocket
Authors: Couture Manon , Das Tapan Kanti , Savard Pierre‐Yves , Ouellet Yannick , Wittenberg Jonathan B , Wittenberg Beatrice A , Rousseau Denis L , Guertin Michel 
Published: March 2003

A putative hemoglobin (Hb) gene, related to those previously characterized in the green alga Chlamydomonas eugametos, the ciliated protozoan Paramecium caudatum, the cyanobacterium Nostoc commune and the bacterium Mycobacterium tuberculosis, was recently discovered in the complete genome sequence of the cyanobacterium Synechocystis PCC 6803. In this paper, we report the purification of Synechocystis Hb and describe some of its salient biochemical and spectroscopic properties. We show that the recombinant protein contains Fe‐protoporphyrin IX and forms a very stable complex with oxygen. The oxygen dissociation rate measured, 0.011 s−1, is among the smallest known and is four orders of magnitude smaller than the rate measured for N. commune Hb, which suggests functional differences between these Hbs. Optical and resonance Raman spectroscopic study of the structure of the heme pocket of Synechocystis Hb reveals that the heme is 6‐coordinate and low‐spin in both ferric and ferrous forms in the pH range 5.5–10.5. We present evidence that His46, predicted to occupy the helical position E10 based on amino‐acid sequence comparison, is involved in the formation of the ferric and ferrous 6‐coordinate low‐spin structures. The analysis of the His46Ala mutant shows that the ferrous form is 5‐coordinate and high‐spin and the ferric form contains a 6‐coordinate high‐spin component in which the sixth ligand is most probably a water molecule. We conclude that the heme pocket of the wild type Synechocystis Hb has a unique structure that requires a histidine residue at the E10 position for the formation of its native structure.
Purification, characterization and molecular modelling of two toxin‐like proteins from the Androctonus australis Hector venom
Authors: Srairi‐Abid Najet , Mansuelle Pascal , Mejri Thouraya , Karoui Habib , Rochat Hervé , Sampieri François , El Ayeb Mohamed 
Published: March 2003

Two toxin‐like proteins (AahTL1 and AahTL3) were purified from the venom of the scorpion Androctonus australis Hector (Aah). AahTL1 and AahTL3 are the first non toxic proteins cross‐reacting with AahI toxins group which indicates that these proteins can be used as a model of vaccins.In order to study structure‐function relationships, their complete amino‐acid sequences (66 residues) were determined, by automated Edman degradation. They show more than 50% of similarity with both AahI and AahIII antimammal toxins. Three‐dimensional structural models of AahTL1 and AahTL3 constructed by homology suggest that the two proteins are structurally similar to antimammal scorpion α‐toxins specific to voltage dependent Na+ channels. The models showed also that amino‐acid changes between potent Aah toxins and both AahTL1 and AahTL3 disrupt the electrostatic potential gradient at their surface preventing their interaction with the receptor, which may explain their non toxicity.