Biochemistry (Moscow) (v.75, #4)

Structural analysis of lipopolysaccharides from Gram-negative bacteria by D. S. Kabanov; I. R. Prokhorenko (383-404).
This review covers data on composition and structure of lipid A, core, and O-polysaccharide of the known lipopolysaccharides from Gram-negative bacteria. The relationship between the structure and biological activity of lipid A is discussed. The data on roles of core and O-polysaccharide in biological activities of lipopolysaccharides are presented. The structural homology of some oligosaccharide sequences of lipopolysaccharides to gangliosides of human cell membranes is considered.
Keywords: lipopolysaccharide (endotoxin); lipooligosaccharide; lipid A; core; O-antigen; gangliosides; cytokines

The 5′-untranslated sequence of tobacco mosaic virus RNA — the so-called omega leader — exhibits features of a translational enhancer of homologous and heterologous mRNAs. The absence of guanylic residues, the presence of multiple trinucleotide CAA repeats in its central region, and the low predictable probability of the formation of an extensive secondary structure of the Watson-Crick type were reported as the peculiarities of the primary structure of the omega leader. In this work we performed chemical and enzymatic probing of the secondary structure of the omega leader. The isolated RNA comprising omega leader sequence was subjected to partial modifications with dimethyl sulfate and diethyl pyrocarbonate and partial hydrolyses with RNase A and RNase V1. The sites and the intensities of the modifications or the cleavages were detected and measured by the primer extension inhibition technique. The data obtained have demonstrated that RNase A, which attacks internucleotide bonds at the 3′ side of pyrimidine nucleotides, and diethyl pyrocarbonate, which modifies N7 of adenines not involved in stacking interactions, weakly affected the core region of omega leader sequence enriched with CAA-repeats, this directly indicating the existence of a stable spatial structure. The significant stability of the core region structure to RNase A and diethyl pyrocarbonate was accompanied by its complete resistance against RNase V1, which cleaves a polyribonucleotide chain involved in Watson-Crick double helices and generally all A-form RNA helices, thus being an evidence in favor of a non-Watson-Crick structure. The latter was confirmed by the full susceptibility of all adenines and cytosines of the omega polynucleotide chain to dimethyl sulfate, which exclusively modifies N1 of adenines and N3 of cytosines not involved in Watson-Crick interactions. Thus, our data have confirmed that (1) the regular (CAA)n sequence characteristic of the core region of the omega leader does form stable secondary structure, and (2) the structure formed is not the canonical double helix of the Watson-Crick type.
Keywords: omega leader of TMV RNA; regular (CAA)n polyribonucleotide; RNA triple helix; chemical modification of RNA; enzymatic cleavage of RNA; primer extension inhibition; diethyl pyrocarbonate; dimethyl sulfate; RNase A; RNase V1

Femtosecond stage of electron transfer in reaction centers of the triple mutant SL178K/GM203D/LM214H of Rhodobacter sphaeroides by A. G. Yakovlev; T. A. Shkuropatova; V. A. Shkuropatova; V. A. Shuvalov (412-422).
Coherent processes in an initial phase of charge transfer in reaction centers (RCs) of the triple mutant S(L178)K/G(M203)D/L(M214)H of Rhodobacter sphaeroides were investigated by difference (light — dark) absorption spectroscopy with 18 fsec time resolution. Electron transfer in the B cofactor branch is activated in this mutant, while the A-branch electron transfer is slowed in comparison with native RCs of Rba. sphaeroides. A bulk of absorption difference spectra was analyzed in the 940–1060 nm range (stimulated emission of excited bacteriochlorophyll dimer P* and absorption of bacteriochlorophyll anions B A and β, where β is a bacteriochlorophyll substituting the native bacteriopheophytin HA) and in the 735–775 nm range (bleaching of the absorption band of the bacteriopheophytin HB in the B-branch) in the −0.1 to 4 psec range of delays with respect to the moment of photoexcitation of P at 870 nm. Spectra were measured at 293 and 90 K. The kinetics of P* stimulated emission at 940 nm shows its decay with a time constant of ∼14 psec at 90 K and ∼18 psec at 293 K, which is accompanied by oscillations with a frequency of ∼150 cm−1. A weak absorption band is found at 1018 nm that is formed ∼100 fsec after excitation of P and reflects the electron transfer from P* to β and/or BA with accumulation of the P+β and/or P+B A states. The kinetics of ΔA at 1018 nm contains the oscillations at ∼150 cm−1 and distinct low-frequency oscillations at 20–100 cm−1; also, the amplitude of the oscillations at 150 cm−1 is much smaller at 293 than at 90 K. The oscillations in the kinetics of the 1018 nm band do not contain a 32 cm−1 mode that is characteristic for native Rba. sphaeroides RCs having water molecule HOH55 in their structure. The ΔA kinetics at 751 nm reflects the electron transfer to HB with formation of the P+H B state. The oscillatory part of this kinetics has the form of a single peak with a maximum at ∼50 fsec completely decaying at ∼200 fsec, which might reflect a reversible electron transfer to the B-branch. The results are analyzed in terms of coherent nuclear wave packet motion induced in the P* excited state by femtosecond light pulses, of an influence of the incorporated mutations on the mutual position of the energy levels of charge separated states, and of the role of water HOH55 in the dynamics of the initial electron transfer.
Keywords: photosynthesis; charge separation; reaction center; wave packet; electron transfer

The effect of dehydration and 2H2O/H2O isotope substitution on electron transport reactions and relaxation of proton-containing groups was studied in chromatophore membranes of Ectothiorhodospira shaposhnikovii. During dehydration (including isotope substitution of hydrate water) of preliminarily dehydrated isolated photosynthetic membranes there was a partial correlation between hydration intervals within which activation of electron transport from high-potential cytochrome c to photoactive bacteriochlorophyll dimer P890 of photosynthetic reaction center and variation of spin-lattice and spin-spin proton relaxation time was observed. Partial correlation between hydration intervals can be considered as evidence of correlation between mobility of non-water proton-containing groups with proton relaxation frequency ∼108 sec−1 with efficiency of electron transfer at the donor side of the chain.
Keywords: electron transport; molecular mobility; NMR; photosynthetic bacteria; Ectothiorhodospira shaposhnikovii

Peroxidase activity of cytochrome bd from Escherichia coli by V. B. Borisov; A. I. Davletshin; A. A. Konstantinov (428-436).
Cytochrome bd from Escherichia coli is able to oxidize such substrates as guaiacol, ferrocene, benzohydroquinone, and potassium ferrocyanide through the peroxidase mechanism, while none of these donors is oxidized in the oxidase reaction (i.e. in the reaction that involves molecular oxygen as the electron acceptor). Peroxidation of guaiacol has been studied in detail. The dependence of the rate of the reaction on the concentration of the enzyme and substrates as well as the effect of various inhibitors of the oxidase reaction on the peroxidase activity have been tested. The dependence of the guaiacol-peroxidase activity on the H2O2 concentration is linear up to the concentration of 8 mM. At higher concentrations of H2O2, inactivation of the enzyme is observed. Guaiacol markedly protects the enzyme from inactivation induced by peroxide. The peroxidase activity of cytochrome bd increases with increasing guaiacol concentration, reaching saturation in the range from 0.5 to 2.5 mM, but then starts falling. Such inhibitors of the ubiquinol-oxidase activity of cytochrome bd as cyanide, pentachlorophenol, and 2-n-heptyl 4-hydroxyquinoline-N-oxide also suppress its guaiacol-peroxidase activity; in contrast, zinc ions have no influence on the enzyme-catalyzed peroxidation of guaiacol. These data suggest that guaiacol interacts with the enzyme in the center of ubiquinol binding and donates electrons into the di-heme center of oxygen reduction via heme b 558, and H2O2 is reduced by heme d. Although the peroxidase activity of cytochrome bd from E. coli is low compared to peroxidases, it might be of physiological significance for the bacterium itself and plays a pathophysiological role for humans and animals.
Keywords: aromatic substrates; cytochrome bd ; Escherichia coli ; oxidation of phenols; peroxidase; respiratory chain; terminal oxidase

Influence of polyelectrolytes of different chemical structure and degree of polymerization on aggregation and denaturation of the oligomeric enzyme glyceraldehyde-3-phosphate dehydrogenase has been studied to ascertain molecular characteristics of the polymer chains providing the efficient prevention of aggregation of the enzyme without drastic changes in its structure and catalytic activity. The best polymers meeting these requirements were found to be hydrophilic high-molecular-weight polyelectrolytes forming stable complexes with the enzyme. The revealed pronounced negative effect of short polymer chains on the enzyme must be taken into account in the design of protein-polyelectrolyte systems by using thoroughly fractionated polymer samples containing no admixture of charged oligomers.
Keywords: glyceraldehyde-3-phosphate dehydrogenase; polysulfoanions; protein-polyelectrolyte complexes; thermoaggregation suppression; differential scanning calorimetry

Biological properties and structure of the lipopolysaccharide of a vaccine strain of Francisella tularensis generated by inactivation of a quorum sensing system gene qseC by A. N. Mokrievich; A. N. Kondakova; E. Valade; M. E. Platonov; G. M. Vakhrameeva; R. Z. Shaikhutdinova; R. I. Mironova; D. Blaha; I. V. Bakhteeva; G. M. Titareva; T. B. Kravchenko; T. I. Kombarova; D. Vidal; V. M. Pavlov; B. Lindner; I. A. Dyatlov; Yu. A. Knirel (443-451).
A knockout mutant with a deletion in a quorum sensing system gene qseC was generated from the vaccine strain Francisella tularensis 15 by site-directed mutagenesis. The variant with the inactivated gene qseC differed from the parental strain in growth rate on solid nutrient medium but had the same growth dynamics in liquid nutrient medium. The mutation abolished almost completely the resistance of the vaccine strain to normal rabbit serum and its ability to survive in macrophages; in addition, the strain lost the residual virulence. A significant phenotypic alteration was observed in the lipopolysaccharide of F. tularensis. Particularly, the mutant strain synthesized no noticeable amount of the lipopolysaccharide with the high-molecular-mass O-polysaccharide, presumably as a result of impairing biosynthesis of the repeating unit, namely, a loss of the ability to incorporate a formyl group, an N-acyl substituent of 4-amino-4,6-dideoxy-D-glucose.
Keywords: Francisella tularensis ; tularemia; quorum sensing; gene qseC ; site-directed mutagenesis; lipopolysaccharide

Molecular characterization and heterologous expression of quinate dehydrogenase gene from Gluconobacter oxydans IFO3244 by A. S. Vangnai; W. Promden; W. De-Eknamkul; K. Matsushita; H. Toyama (452-459).
The quinate dehydrogenase (QDH) from Gluconobacter oxydans IFO3244 exhibits high affinity for quinate, suggesting its application in shikimate production. Nucleotide sequence analysis of the qdh gene revealed a full-length of 2475-bp encoding an 824-amino acid protein. The qdh gene has the unusual TTG translation initiation codon. Conserved regions and a signature sequence for the quinoprotein family were observed. Phylogenetic analysis demonstrated relatedness of QDH from G. oxydans to other quinate/shikimate dehydrogenases with the highest similarity (56%) with that of Acinetobacter calcoaceticus ADP1 and lower similarity (36%) with a membrane-bound glucose dehydrogenase of Escherichia coli. The function of the gene coding for QDH was confirmed by heterologous gene expression in pyrroloquinoline quinone-synthesizing Pseudomonas putida HK5.
Keywords: quinate dehydrogenase gene; heterologous gene expression; Gluconobacter oxydans ; pyrroloquinoline quinone

We report the acid tolerance response and changes in the level of protein expression of probiotic Lactobacillus reuteri subjected to transient (1.5 h) acid stress at pH 3.0. Sixteen acid-responsive proteins were identified by peptide mass fingerprinting including members of five broad functional categories: metabolism, transcription/translation, DNA replication/repair, transport and binding proteins, and pH homeostasis and stress responses. This work can provide some new and relevant information on the inducible mechanisms underlying the capacity of probiotic L. reuteri to tolerate acid stress.
Keywords: Lactobacillus reuteri ; acid stress response; two-dimensional gel electrophoresis (2-DE); matrix-assisted laser-desorption-ionization time-of-flight-mass spectrometry (MALDI-TOF MS)

Expression and purification of the fusion protein HMGB1Abox-TMD1, a novel HMGB1 antagonist by Yuan Li; Wei Gong; Li Zhang; Gang Huang; Jialin Li; Xiaodong Shen; Fengtian He (466-471).
High mobility group box chromosomal protein 1 (HMGB1) is a lethal mediator of systemic inflammation, and its A box domain is isolated as an antagonist of HMGB1. To enhance its expression level and its anti-HMGB1 effect, the A box cDNA was coupled with the sequence encoding lectin-like domain of thrombomodulin (TMD1). The fusion DNA fragment was ligated into the prokaryotic expression vector pQE-80L to construct the recombinant plasmid pQE80L-A/TMD1. The plasmid was then transformed into Escherichia coli DH5α, and the recombinant fusion protein A/TMD1 was expressed at 37°C for 4 h, with induction by IPTG at the final concentration of 0.2 mM. The expression level of the fusion protein was up to 40% of the total cellular protein. The fusion protein was purified by Ni-NTA chromatography and the purity was about 95%. After passing over a polymyxin B column to remove any contaminating lipopolysaccharides, the purified protein was tested for its anti-inflammatory activity. Our data show that A/TMD1 significantly inhibits HMGB1-induced TNF-α release and might be useful in treating HMGB1-elevated sepsis.
Keywords: fusion protein; high mobility group box chromosomal protein 1; thrombomodulin

Characterization of glycoprotein E C-End of West Nile virus and evaluation of its interaction force with αVβ3 integrin as putative cellular receptor by M. V. Bogachek; B. N. Zaitsev; S. K. Sekatskii; E. V. Protopopova; V. A. Ternovoi; A. V. Ivanova; A. V. Kachko; V. A. Ivanisenko; G. Dietler; V. B. Loktev (472-480).
Recombinant polypeptide containing the 260–466 amino acid sequence of West Nile virus (WNV) strain LEIV-Vlg99-27889-human glycoprotein E (gpE, E260–466) was constructed. Immunochemical similarity between the E260–466 and gpE of WNV was proven by enzyme immunoassay (EIA), immunoblot, competitive EIA, hemagglutination inhibition, and neutralization tests using polyclonal and monoclonal antibodies against the viral gpE and recombinant E260–466. Polypeptide E260–466 induced formation of virus neutralizing and cross-reactive antibodies that were interactive with various epitopes of this recombinant protein. It is shown by evaluation of the interaction of E260–466 with one of the proposed cell receptors of WNV that average E260–466-αVβ3 integrin-specific interaction force measured using atomic force spectroscopy was 80 and 140 pN for single and double interactions, correspondingly. Taken together with previously described interaction between laminin-binding protein (LBP) and WNV gpE domain II, it is proposed that WNV gpE can interact specifically with two cellular proteins (LBP and αVβ3 integrin) during virus entry.
Keywords: West Nile virus; glycoprotein E; atomic force spectroscopy; αVβ3 integrin

Production of bioactive human hemangiopoietin in Escherichia coli by Jie Zhang; Jian-Feng Li; Shuang-Quan Zhang (481-485).
To devise an efficient approach for production of human hemangiopoietin (hHAPO), the gene of hHAPO was synthesized and subcloned into the pSUMO vector with a SUMO tag at the N-terminus. The expression construct was then transformed into the expression strain E. coli BL21(DE3). The fusion protein was expressed in soluble form and identified by SDS-PAGE and Western blotting. The fusion protein was purified to 90% purity by metal chelate chromatography with a yield of 45 mg per liter fermentation culture. The SUMO tag was removed by cleavage with SUMO protease at room temperature for 1 h, and the hHAPO was then re-purified by the metal chelate chromatography. Finally, about 21 mg hHAPO was obtained from 1 liter of fermentation culture with no less than 95% purity. The recombinant hHAPO significantly stimulated the proliferation of human umbilical vein endothelial cells.
Keywords: E. coli ; fermentation; hemangiopoietin; Ni-NTA; purification; SUMO

Effect of trifluoroethanol on native and acid-induced states of glucose oxidase from Aspergillus niger by B. Ahmad; S. K. Haq; A. Varshney; A. A. Moosavi-Movahedi; R. H. Khan (486-493).
We have studied the effect of trifluoroethanol (TFE) on the native (pH 7.0), acid unfolded (pH 2.6), and molten globule (pH 1.4) states of glucose oxidase (GOX) by circular dichroism and fluorescence spectroscopy. In the presence of 50% TFE, at pH 7.0 and 2.6, GOX exhibited a transition from native coiled-coil and acid unfolded state to non-associating α-helical state. Interestingly, at pH 1.4, 15% TFE induced the formation of β-structured intermediate by loss of 1-anilino-8-naphthalenesulfonate binding site and almost all tertiary contacts. The β-structured intermediate converted into open helical conformation on further addition of TFE.
Keywords: glucose oxidase; molten globule; coiled-coil state; acid unfolded state; trifluoroethanol

Energy metabolism of Leishmania donovani parasite has been investigated under conditions imitating intralysosomal-like environment in the host organism. Trans-plasma membrane electron transport and oxygen uptake were inhibited progressively when promastigote cells were exposed to pH 5.5 and 37°C. A special feature of the respiratory chain in amastigote was the absence of complex I, II, and IV. When L. donovani was grown at pH 5.5 and 37°C, the acid excretory product succinate was increased in comparison to cells grown at pH 7.5 and 24°C. The findings of this study showed that the amastigote form catabolized fatty acid to excrete succinic acid when oxidative phosphorylation was impaired. Amastigote mitochondria failed to generate membrane potential by oxidizable substrates. On the other hand, the amastigote cell showed absorbance change of safranine O when fatty acid was the oxidizable substrate. The safranine signal was completely reversed by valinomycin, carbonyl cyanide 4-(trifluromethoxy)phenylhydrazone, malonate, and oxaloacetate. Our data suggest that the generation of metabolic energy from succinate/H+ efflux will contribute to energy requiring process of amastigote significantly. On the basis of these results, we conclude that due to absence of oxidative phosphorylation in amastigotes, energy linked functions in amastigotes might occur through fumarate reduction leading to ΔpH generation by succinate excretion.
Keywords: Leishmania donovani promastigote and amastigote; mitochondria; differentiation; stage specific

A new metal complex, Fe(Sal2dienNO3·H2O) (where Sal is salicylaldehyde and dien is diethylenetriamine), has been synthesized and characterized. The interactions between the Fe(III) complex and calf thymus DNA has been investigated using UV and fluorescence spectra, viscosity, thermal denaturation, and molecular modeling. The cleavage reaction on plasmid DNA has been monitored by agarose gel electrophoresis. The experimental results show that the mode of binding of the complex to DNA is classical intercalation and the complex can cleave pBR322 DNA.
Keywords: DNA binding; Fe(Sal2dienNO3·H2O); Fe(III) complex; calf thymus DNA

A putative β-glucosidase gene from the genome of Bacillus halodurans C-125 was expressed in E. coli under the regulation of T7lac promoter. On induction with isopropyl-β-D-1-thiogalactopyranoside, the enzyme expressed at ∼40% of the cell protein producing 238 mg/liter culture. With increase in culture cell density to A 600 12 in auto-inducing M9NG medium, β-glucosidase production increased 3-fold. Approximately 70% of the expressed enzyme was in a soluble form, while the rest was in an insoluble fraction of the cell lysate. The soluble and active form of the expressed enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography to a purity >98%. The mass of the enzyme as determined by MALDI-TOF mass spectrometry was 51,601 Da, which is nearly the same as the calculated value. Phylogenetic analysis of the β-glucosidase of B. halodurans was found to cluster with members of the genus Bacillus. Temperature and pH optima of the enzyme were found to be 45°C and 8.0, respectively, under the assay conditions. K m and k cat against p-nitrophenyl-β-D-glucopyranoside were 4 mM and 0.75 sec−1, respectively. To our knowledge, this is the first report of high-level expression and characterization of a β-glucosidase from B. halodurans.
Keywords: expression; auto-inducing medium; MALDI-TOF analysis; β-glucosidase; Bacillus halodurans ; E. coli

A powerful hybrid puc operon promoter tightly regulated by both IPTG and low oxygen level by Zongli Hu; Zhiping Zhao; Yu Pan; Yun Tu; Guoping Chen (519-525).
Rhodobacter sphaeroides has been intensively studied and provides an excellent model for studying both photo-synthesis and membrane development. The photosynthetic apparatus (LH2 and LH1-RC complexes) can be synthesized in large scale and integrated into the intracytoplasmic membrane system under specific conditions, which thus provides us insight to utilize the puc or(and) puf operon to heterologously express recombinant proteins in the intracytoplasmic membrane using Rb. sphaeroides as a novel expression system. However, basal level of expression of puc and puf promoter is uncontrolled. We report the construction of LH2 polypeptide expression vector that contains a reengineered lacI q-puc promoter-lac operator hybrid promoter, which allows the puc operon to be regulated by both IPTG and low oxygen level. Synthesis of LH2 complexes was completely repressed in the absence of isopropyl β-D-thiogalactoside (IPTG), and the degree of induction was controlled by varying the concentration of IPTG. The optimal concentration of IPTG was determined. SDS-PAGE and Western blot were employed for further analysis. Our results suggest that the reengineered hybrid promoter is efficient to tightly regulate the expression of the puc operon, and our strategy can open up a new approach in the study of the membrane protein expression system.
Keywords: LH2; hybrid promoter; IPTG; Rhodobacter sphaeroides

The structural gene ldhL for a thermostable L-(+)-lactate dehydrogenase was cloned from Thermoanaerobacter ethanolicus JW200. The nucleotide sequence of the ldhL gene was determined and shown to have the capacity to encode a protein of 311 amino acids (33.5 kDa). By 5′-RACE analysis, the ldhL transcription start point was confirmed to be derived from the −10 region closest to the initiation codon. The enzyme was overexpressed in Escherichia coli and purified to homogeneity by nickel-affinity chromatography. It was shown to be allosteric in the presence of fructose-1,6-bisphosphate. The optimum pH and temperature for the enzyme were 5.8 and 60°C in the pyruvate reduction and 7.0 and 70°C in the lactate oxidation reaction, respectively. The kinetic parameters K m,app and kcat,app for pyruvate were 0.18 mM and 520 U/mg, respectively, and in the absence of fructose-1,6-bisphosphate, a 1.2-fold increase in K m,app and a 16-fold decrease in k cat,app were determined. The K m,app and k cat,app values for lactate were 60 mM and 0.58 U/mg, respectively, and they were not affected by fructose-1,6-bisphosphate. The enzyme was greatly inhibited by Zn2+, Ag+, Cu2+, Fe3+, and Pb3+. The extreme thermostability of the enzyme was reflected in its unaltered activity over 5 h at 70°C.
Keywords: L-(+)-lactate dehydrogenase; Thermoanaerobacter ethanolicus JW200; allosteric; thermostable; transcription start point

Glycomics by G. Wiederschain (531-531).