European Journal of Biochemistry

The core structure of the lipopolysaccharide (LPS) isolated from a rough strain of the phytopathogenic bacterium Pseudomonas syringae pv. phaseolicola, GSPB 711, was investigated by sugar and methylation analyses, Fourier transform ion-cyclotron resonance ESI MS, and one- and two-dimensional 1H-, 13C- and 31P-NMR spectroscopy. Strong alkaline deacylation of the LPS resulted in two core-lipid A backbone undecasaccharide pentakisphosphates in the ratio approximately 2.5 : 1, which corresponded to outer core glycoforms 1 and 2 terminated with either L-rhamnose or 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), respectively...

Retinoid receptors are ligand-dependent transcription factors belonging to the nuclear receptor superfamily. Retinoic acid (RARalpha, beta, gamma) and retinoid X (RXRalpha, beta, gamma) receptors mediate the retinoid/rexinoid signal to the transcriptional machineries by interacting at the first level with coactivators or corepressors, which leads to the recruitment of enzymatically active noncovalent complexes at target gene promoters. It has been shown that the interaction of corepressors with nuclear receptors involves conserved LXXI/HIXXXI/L consensus sequences termed corepressor nuclear receptor (CoRNR) boxes...

The NMR solution structures of NTX-1 (PDB code 1W6B and BMRB 6288), a long neurotoxin isolated from the venom of Naja naja oxiana, and the molecular dynamics simulation of these structures are reported. Calculations are based on 1114 NOEs, 19 hydrogen bonds, 19 dihedral angle restraints and secondary chemical shifts derived from 1H to 13C HSQC spectrum. Similar to other long neurotoxins, the three-finger like structure shows a double and a triple stranded beta-sheet as well as some flexible regions, particularly at the tip of loop II and the C-terminal tail...

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue...

RNA reprogramming represents a new concept in correcting genetic defects at the RNA level. However, for the technique to be useful for therapy, the level of reprogramming must be appropriate. To improve the efficiency of group I ribozyme-mediated RNA reprogramming, when using the Tetrahymena ribozyme, regions complementary to the target RNA have previously been extended in length and accessible sites in the target RNAs have been identified. As an alternative to the Tetrahymena model ribozyme, the DiGIR2 group I ribozyme, derived from a mobile group I intron in rDNA of the myxomycete Didymium iridis, represents a new and attractive tool in RNA reprogramming...

Bacterial RNA polymerase (RNAP) is the central enzyme of gene expression that is responsible for the synthesis of all types of cellular RNAs. The process of transcription is accompanied by complex structural rearrangements of RNAP. Despite the recent progress in structural studies of RNAP, detailed mechanisms of conformational changes of RNAP that occur at different stages of transcription remain unknown. The goal of this work was to obtain novel ligands to RNAP which would target different epitopes of the enzyme and serve as specific probes to study the mechanism of transcription and conformational flexibility of RNAP...

Loss of cannabinoid receptors (CB1) occurs prior to neurodegeneration in Huntington's disease (HD). The levels and distribution of CB1 RNA were equivalent in 3-week-old mice regardless of genotype demonstrating that the specific factors and appropriate chromatin structure that lead to the transcription of CB1 were present in the striatum of young R6/2 and R6/1 transgenic HD mice. The expression of the mutant HD transgene led progressively to decreased steady-state levels of CB1 mRNA in neurons of the lateral striatum, which was dependent on the size of the CAG repeat and relative expression of the gene encoding mutant huntingtin (HD)...

Incubation of an NAD+-dependent succinic semialdehyde dehydrogenase from bovine brain with 4-dimethylaminoazobenzene-4-iodoacetamide (DABIA) resulted in a time-dependent loss of enzymatic activity. This inactivation followed pseudo first-order kinetics with a second-order rate constant of 168 m(-1).min(-1). The spectrum of DABIA-labeled enzyme showed a characteristic peak of the DABIA alkylated sulfhydryl group chromophore at 436 nm, which was absent from the spectrum of the native enzyme. A linear relationship was observed between DABIA binding and the loss of enzyme activity, which extrapolates to a stoichiometry of 8...

The c-jun belongs to the family of proto-oncogenes and encodes for the protein Jun, a component of transcription factor AP-1 involved in regulation of the expression of genes indispensable for cell proliferation and differentiation. While the role of c-jun in the regulation of such genes has been well examined, the regulation of c-jun in proliferating cells is not fully understood. We have earlier reported that the -148 to -124 region of c-jun is involved in the positive regulation of c-jun transcription, and interacts with a positive regulatory factor (rat liver jun regulatory protein; RLjunRP) present in rat liver...

Cytosolic 5'-nucleotidase/phosphotransferase specific for 6-hydroxypurine monophosphate derivatives (cN-II), belongs to a class of phosphohydrolases that act through the formation of an enzyme-phosphate intermediate. Sequence alignment with members of the P-type ATPases/L-2-haloacid dehalogenase superfamily identified three highly conserved motifs in cN-II and other cytosolic nucleotidases. Mutagenesis studies at specific amino acids occurring in cN-II conserved motifs were performed. The modification of the measured kinetic parameters, caused by conservative and nonconservative substitutions, suggested that motif I is involved in the formation and stabilization of the covalent enzyme-phosphate intermediate...

With the aim of extending our knowledge on the reaction pathways of Zn-metallothionein (MT) and apo-MT species in the presence of Hg(II), we monitored the titration of Zn7-MT, Zn4-alphaMT and Zn3-betaMT proteins, at pH 7 and 3, with either HgCl2 or Hg(ClO4)2 by CD and UV-vis spectroscopy. Detailed analysis of the optical data revealed that standard variables, such as the pH of the solution, the binding ability of the counter-ion (chloride or perchlorate), and the time elapsed between subsequent additions of Hg(II) to the protein, play a determinant role in the stoichiometry, stereochemistry and degree of folding of the Hg-MT species...

ATP-dependent Lon proteases belong to the superfamily of AAA+ proteins. Until recently, the identity of the residues involved in their proteolytic active sites was not elucidated. However, the putative catalytic Ser-Lys dyad was recently suggested through sequence comparison of more than 100 Lon proteases from various sources. The presence of the catalytic dyad was experimentally confirmed by site-directed mutagenesis of the Escherichia coli Lon protease and by determination of the crystal structure of its proteolytic domain...

The addition of glucose to Saccharomyces cerevisiae cells causes reprogramming of gene expression. Glucose is sensed by membrane receptors as well as (so far elusive) intracellular sensing mechanisms. The availability of four yeast strains that display different hexose uptake capacities allowed us to study glucose-induced effects at different glycolytic rates. Rapid glucose responses were observed in all strains able to take up glucose, consistent with intracellular sensing. The degree of long-term responses, however, clearly correlated with the glycolytic rate: glucose-stimulated expression of genes encoding enzymes of the lower part of glycolysis showed an almost linear correlation with the glycolytic rate, while expression levels of genes encoding gluconeogenic enzymes and invertase (SUC2) showed an inverse correlation...

We have cloned, over-expressed and purified enolase from Plasmodium falciparum strain NF54 in Escherichia coli in active form, as an N-terminal His6-tagged protein. The sequence of the cloned enolase from the NF54 strain is identical to that of strain 3D7 used in full genome sequencing. The recombinant enolase (r-Pfen) could be obtained in large quantities (approximately 50 mg per litre of culture) in a highly purified form (> 95%). The purified protein gave a single band at approximately 50 kDa on SDS/PAGE...

The extremely heat-stable 5'-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi-Bi mechanism and phosphate binding precedes nucleoside binding in the phosphorolytic direction. 5'-Methylthioadenosine phosphorylase from Pyrococcus furiosus is a hexameric protein with five cysteine residues per subunit...

The role of vertebrate histone proteins or histone derived peptides as innate immune effectors has only recently been appreciated. In this study, high levels of core histone proteins H2A, H2B, H3 and H4 were found in hemocytes from the Pacific white shrimp, Litopenaeus vannamei. The proteins were identified by in-gel digestion, mass spectrometry analysis, and homology searching. The L. vannamei histone proteins were found to be highly homologous to histones of other species. Based on this homology, histone H2A was cloned and its N-terminus was found to resemble the known antimicrobial histone peptides buforin I, parasin, and hipposin...

High-pressure NMR experiments performed on the histidine-containing phosphocarrier protein (HPr) from Staphylococcus carnosus have shown that residue Ile14, which is located in the active-centre loop, exhibits a peculiarly small pressure response. In contrast, the rest of the loop shows strong pressure effects as is expected for typical protein interaction sites. To elucidate the structural role of this residue, the mutant protein HPr(I14A), in which Ile14 is replaced by Ala, was produced and studied by solution NMR spectroscopy...

Human kynurenine aminotransferase I/glutamine transaminase K (hKAT-I) is an important multifunctional enzyme. This study systematically studies the substrates of hKAT-I and reassesses the effects of pH, Tris, amino acids and alpha-keto acids on the activity of the enzyme. The experiments were comprised of functional expression of the hKAT-I in an insect cell/baculovirus expression system, purification of its recombinant protein, and functional characterization of the purified enzyme. This study demonstrates that hKAT-I can catalyze kynurenine to kynurenic acid under physiological pH conditions, indicates indo-3-pyruvate and cysteine as efficient inhibitors for hKAT-I, and also provides biochemical information about the substrate specificity and cosubstrate inhibition of the enzyme...

Bifidobacterium bifidum is a useful probiotic agent exhibiting health-promoting properties and contains d-aspartate as an essential component of the cross-linker moiety in the peptidoglycan. To help understand D-aspartate biosynthesis in B. bifidum NBRC 14252, aspartate racemase, which catalyzes the racemization of D- and L-aspartate, was purified to homogeneity and characterized. The enzyme was a monomer with a molecular mass of 27 kDa. This is the first report showing the presence of a monomeric aspartate racemase...

Saccharomyces cerevisiae is a widely used host in the production of therapeutic peptides and proteins. Here we report the identification of a novel endoprotease in S. cerevisiae. It is encoded by the CYM1 gene and is specific for the C-terminus of basic residues of heterologously expressed peptides. Gene disruption of CYM1 not only reduced the intracellular proteolysis, but also enhanced the secretion of heterologously expressed peptides such as growth hormone, pro-B-type natriuretic peptide and pro-cholecystokinin...