Biochemistry (Moscow) (v.73, #1)

Cysteine proteinases of microorganisms and viruses by G. N. Rudenskaya; D. V. Pupov (1-13).
This review considers properties of secreted cysteine proteinases of protozoa, bacteria, and viruses and presents information on the contemporary taxonomy of cysteine proteinases. Literature data on the structure and physicochemical and enzymatic properties of these enzymes are reviewed. High interest in cysteine proteinases is explained by the discovery of these enzymes mostly in pathogenic organisms. The role of the proteinases in pathogenesis of several severe diseases of human and animals is discussed.
Keywords: cysteine proteinases; properties; protozoa; bacteria; viruses

Comparative studies on the interaction of the membrane-bound and detergent-solubilized forms of the enzyme in the fully reduced state with carbon monoxide at room temperature have been carried out. CO brings about a bathochromic shift of the heme d band with a maximum at 644 nm and a minimum at 624 nm, and a peak at 540 nm. In the Soret band, CO binding to cytochrome bd results in absorption decrease and minima at 430 and 445 nm. Absorption perturbations in the Soret band and at 540 nm occur in parallel with the changes at 630 nm and reach saturation at 3–5 μM CO. The peak at 540 nm is probably either β-band of the heme d-CO complex or part of its split α-band. In both forms of cytochrome bd, CO reacts predominantly with heme d. Addition of high CO concentrations to the solubilized cytochrome bd results in additional spectral changes in the γ-band attributable to the reaction of the ligand with 10–15% of low-spin heme b 558. High-spin heme b 595 does not bind CO even at high concentrations of the ligand. The apparent dissociation constant values for the heme d-CO complex of the membrane-bound and detergent-solubilized forms of the fully reduced enzyme are about 70 and 80 nM, respectively.
Keywords: respiratory chain; terminal oxidase; cytochrome bd ; ligand binding; carbon monoxide; energy conservation; Escherichia coli

In the present study, a novel structural motif of proteins referred to as the φ-motif is considered, and two novel structural trees in which the φ-motif is taken as the root structure have been constructed. The simplest φ-motif is formed by three adjacent β-strands connected by loops and packed in one β-sheet so that its overall fold resembles the Greek letter φ. Construction of the structural trees and modeling of folding pathways have shown that all structures of the protein superfamilies can be obtained by stepwise addition of α-helices and/or β-strands to the root φ-motif taking into account a restricted set of rules inferred from known principles of protein structure. The structural trees are a good tool for structure comparison, structural classification of proteins, as well as for searching for all possible protein folds and folding pathways.
Keywords: protein structure similarity; classification; modeling; protein folding

Role of P-glycoprotein in evolution of populations of chronic myeloid leukemia cells treated with imatinib by T. P. Stromskaya; E. Yu. Rybalkina; S. S. Kruglov; T. N. Zabotina; E. B. Mechetner; A. G. Turkina; A. A. Stavrovskaya (29-37).
Imatinib mesylate (imatinib) is a new generation preparation that is now successfully used for treatment of cancer, particularly for chemotherapy of chronic myeloid leukemia (CML). Imatinib inhibits the activity of chimeric kinase BCR-ABL, which is responsible for the development of CML. The goal of this study was to investigate the role of a multidrug resistance protein, P-glycoprotein (Pgp), in the evolution of CML treated with imatinib. We demonstrate here that although imatinib is a substrate for Pgp, cultured CML cells (strain K562/i-S9), overexpressing active Pgp, do not exhibit imatinib resistance. Studies of CML patients in the accelerated phase have shown variations in the number of Pgp-positive cells (Pgp+) among individual patients treated with imatinib. During treatment of patients with imatinib for 6–12 months, the number of Pgp-positive cells significantly increased in most patients. The high number of Pgp+ cells remained in patients at least for 4.5 years and correlated with active Rhodamine 123 (Rh123) efflux. Such correlation was not found in the group of imatinib-resistant patients examined 35–60 months after onset of imatinib therapy: cells from the imatinibresistant patients exhibited efficient Rh123 efflux irrespectively of Pgp expression. We also compared the mode of Rh123 efflux by cells from CML patients who underwent imatinib treatment for 6–24 months and the responsiveness of patients to this therapy. There were significant differences in survival of patients depending on the absence or the presence of Rh123 efflux. In addition to Pgp, patients’ cells expressed other transport proteins of the ABC family. Our data suggest that treatment with imatinib causes selection of leukemic stem cells characterized by expression of Pgp and other ABC transporters.
Keywords: P-glycoprotein; chronic myeloid leukemia; imatinib; tyrosine kinase inhibitor; BCR-ABL

Identification of a novel nuclear-localized adenylate kinase from Drosophila melanogaster by Geng Meng; Ruitong Zhai; Bin Liu; Xiaofeng Zheng (38-43).
As a step to further understand the role of adenylate kinase (AK) in the energy metabolism network, we identified, purified, and characterized a previously underscribed adenylate kinase in Drosophila melanogaster. The cDNA encodes a 175-amino acid protein, which shows 47.85% identity in 163 amino acids to human AK6. The recombinant protein was successfully expressed in Escherichia coli BL21 (DE3) strain. Characterization of this protein by enzyme activity assay showed adenylate kinase activity. AMP and CMP were the preferred substrates, and UMP can also be phosphorylated to some extent, with ATP as the best phosphate donor. Subcellular localization study showed a predominantly nuclear localization. Therefore, based on the substrate specificity, the specific nuclear localization in the cell, and the sequence similarity with human AK6, we named this novel adenylate kinase identified from the fly DAK6.
Keywords: cloning; protein expression; adenylate kinase activity; nuclear localization

Complete sequencing of potato virus X new strain genome and construction of viral vector for production of target proteins in plants by N. V. Ravin; E. S. Mardanova; R. Yu. Kotlyarov; V. K. Novikov; J. G. Atabekov; K. G. Skryabin (44-49).
The complete nucleotide sequence of the genome of a new potato virus X (PVX) strain Tula isolated by us has been determined. Based on comparison of the PVX Tula nucleotide sequence with the sequences of 12 other PVX strains, this strain was assigned to the European cluster of PVX strains. Phylogenetic analysis revealed the same phylogeny for both full genome sequences and nucleotide sequences of polymerase and coat protein genes, suggesting that the PVX evolution did not involve recombination between different strains. The full-size cDNA copy of the PVX Tula genome was cloned and the accumulation of the viral coat protein in infected Nicotiana benthamiana was shown to be about twofold higher than for the PVX strain UK3. Based on the PVX Tula genome, a new vector which contained the target gene instead of the removed triple transport gene block and the coat protein gene has been constructed for expression of target proteins in plants. The productivity of the new vector was about 1.5–2-fold higher than the productivity of the vector of the same structure based on the standard PVX strain genome. The new viral vector can be used for superproduction of recombinant proteins in plants.
Keywords: potato virus X; plants as biofactories; viral vector

Oligomerization of the potato virus X 25-kD movement protein by A. D. Leshchiner; E. A. Minina; D. V. Rakitina; V. K. Vishnichenko; A. G. Solovyev; S. Yu. Morozov; N. O. Kalinina (50-55).
A 25-kD movement protein (25K protein) encoded by the first gene of the potexvirus Potato virus X triple gene block of transport genes is essential for the viral movement in infected plants. The 25K protein belongs to superfamily 1 of NTPase/helicases and exhibits in vitro RNA helicase, Mg2+-dependent NTPase, and RNA-binding activities. In the present work, the ability of 25K protein for homologous interactions was studied using the yeast two-hybrid system, protein chemical cross-linking in the presence of glutaraldehyde, far-Western blotting, and ultracentrifugation in sucrose density gradients. The 25K protein was shown to form homodimers and homooligomers. Sites of homologous protein-protein interactions were found in both the N- and C-terminal portions of the protein.
Keywords: potato virus X; movement protein; oligomerization; yeast two-hybrid system; glutaraldehyde

Molecular modeling studies of substrate binding by penicillin acylase by G. G. Chilov; O. V. Stroganov; V. K. Švedas (56-64).
Molecular modeling has revealed intimate details of the mechanism of binding of natural substrate, penicillin G (PG), in the penicillin acylase active center and solved questions raised by analysis of available X-ray structures, mimicking Michaelis complex, which substantially differ in the binding pattern of the PG leaving group. Three MD trajectories were launched, starting from PDB complexes of the inactive mutant enzyme with PG (1FXV) and native penicillin acylase with sluggishly hydrolyzed substrate analog penicillin G sulfoxide (1GM9), or from the complex obtained by PG docking. All trajectories converged to a similar PG binding mode, which represented the near-to-attack conformation, consistent with chemical criteria of how reactive Michaelis complex should look. Simulated dynamic structure of the enzyme-substrate complex differed significantly from 1FXV, resembling rather 1GM9; however, additional contacts with residues bG385, bS386, and bN388 have been found, which were missing in X-ray structures. Combination of molecular docking and molecular dynamics also clarified the nature of extremely effective phenol binding in the hydrophobic pocket of penicillin acylase, which lacked proper explanation from crystallographic experiments. Alternative binding modes of phenol were probed, and corresponding trajectories converged to a single binding pattern characterized by a hydrogen bond between the phenol hydroxyl and the main chain oxygen of bS67, which was not evident from the crystal structure. Observation of the trajectory, in which phenol moved from its steady bound to pre-dissociation state, mapped the consequence of molecular events governing the conformational transitions in a coil region a143-a146 coupled to substrate binding and release of the reaction products. The current investigation provided information on dynamics of the conformational transitions accompanying substrate binding and significance of poorly structured and flexible regions in maintaining catalytic framework.
Keywords: penicillin acylase; substrate binding; molecular modeling; conformational flexibility

Content and chain lengths of inorganic polyphosphates (polyP) as well as exopolyphosphatase activities were compared in cytosol and mitochondria of the yeast Saccharomyces cerevisiae during growth on glucose or ethanol under phosphate surplus. PolyP metabolism in cytosol and mitochondria was substantially dependent upon the carbon source. Acid-soluble polyP accumulated mainly in cytosol using either glucose or ethanol. The level of the accumulation was lower during growth on ethanol compared to that on glucose. Increase in polyP content in mitochondria was observed during growth on glucose, but not on ethanol. In cytosol the activity of exopolyphosphatase PPN1 was increased and the activity of exopolyphosphatase PPX1 was decreased independently of the carbon source under phosphate surplus conditions. Growth on ethanol caused exopolyphosphatase PPN1 to appear in the soluble mitochondrial fraction, while during growth on glucose only exopolyphosphatase PPX1 was present in this fraction.
Keywords: inorganic polyphosphates (polyP); exopolyphosphatase; cytosol; mitochondria; phosphate surplus; yeast; Saccharomyces cerevisiae

Detection of target genes of FOXA transcription factors involved in proliferation control by L. O. Bryzgalov; N. I. Ershov; D. Yu. Oshchepkov; V. I. Kaledin; T. I. Merkulova (70-75).
To reveal the mechanism of tumor-suppressing activity of FOXA proteins in liver, a search for potential target genes of these transcription factors involved in proliferation control was carried out. In the first step, we have used data from the literature concerning gene expression in mouse liver (high content of FOXA proteins) and kidney (FOXA expression is absent) obtained by hybridization on microchips. A search for FOXA binding sites in regulatory regions of forty differentially expressing genes involved in proliferation control was carried out using the computer method SITECON. Eleven genes containing clusters of potential FOXA sites incorporating 3–6-fold repeats of TTTG were revealed. The FOXA-specific interaction with such microsatellite sites was confirmed by gel-retardation technique using the GST-fused protein containing the DNA-binding domain of FOXA2. Six genes containing clusters of confirmed binding sites—Cul2, Cdc73, Ptk, Pdcd, Creb, and Ppp2r5d—were selected. The effect of hepatocarcinogen orthoaminoazotoluene (OAT), which lowers the FOXA activity, on expression of these genes was studied by the real-time PCR. OAT was shown to increase sharply the level of mRNA of the Cul2 and Cdc73 genes.
Keywords: FOXA transcription factors; target genes; proliferation control; liver

The effects of lipoic acid on intensity of free radical reactions, citrate content, and aconitate hydratase during myocardial ischemia have been investigated. Treatment with lipoic acid normalized biochemiluminescence parameters and citrate level, which were increased in the myocardial pathology. Treatment with lipoic acid also increased specific activity of aconitate hydratase, which was decreased in myocardium and blood of animals with myocardial ischemia. Administration of lipoic acid decreased DNA fragmentation observed during myocardial ischemia. The data suggest that lipoic acid can be effectively used as a cardioprotector preventing the development of free radical oxidation during myocardial ischemia.
Keywords: myocardial ischemia; lipoic acid; citrate; aconitate hydratase

Investigation of enzymatic degradation of pectin polysaccharides under limiting conditions by N. Yu. Selivanov; I. V. Sorokina; O. G. Selivanova; O. I. Sokolov; V. V. Ignatov (80-86).
The dynamics of changes in spectra of oligosaccharide fragments formed during enzymatic degradation of plant pectins at low enzyme/substrate ratio was studied. It is shown that degradation of deesterified pectin molecules is a discrete and determined process manifested in establishment of a stable polysaccharide spectrum. It is noted that introduction of chemical modifications into the polysaccharide substrate structure preserves the discreteness of the polymer molecule fragmentation but changes the spectrum of formed oligosaccharide fragments. It is supposed that degradation is defined by the spatial (three-dimensional) organization of the polysaccharide molecule.
Keywords: plant polysaccharides; pectin; oligosaccharides; enzymatic degradation; pectinase

An essential tryptophan residue in alkaline phosphatase from pearl oyster (Pinctada fucata) by Li-Ping Xie; Guang-Rui Xu; Wei-Zhong Cao; Jin Zhang; Rong-Qing Zhang (87-91).
Alkaline phosphatases are ubiquitous enzymes found in most species including the pearl oyster, Pinctada fucata, where it is presumably involved in nacreous biomineralization processes. In the present study, we have purified alkaline phosphatases from the pearl oyster and modified the tryptophan residues using N-bromosuccinimide (NBS). We show that the resulting inactivation of purified alkaline phosphatase by NBS is dependent on modification of only one of five tryptophan residues in the enzyme. Substrate protection experiments showed that the tryptophan residue was not located at the substrate-binding site but was involved in the catalytic activity.
Keywords: alkaline phosphatase; Pinctada fucata ; chemical modification; kinetics

Mechanism of inhibition of catalase by nitro and nitroso compounds by V. Yu. Titov; Yu. M. Petrenko; A. F. Vanin (92-96).
Dinitrosyl iron complexes (DNIC) with thiolate ligands and S-nitrosothiols, which are NO and NO+ donors, share the earlier demonstrated ability of nitrite for inhibition of catalase. The efficiency of inhibition sharply (by several orders in concentration of these agents) increases in the presence of chloride, bromide, and thiocyanate. The nitro compounds tested—nitroarginine, nitroglycerol, nitrophenol, and furazolidone—gained the same inhibition ability after incubation with ferrous ions and thiols. This is probably the result of their transformation into DNIC. None of these substances lost the inhibitory effect in the presence of the well known NO scavenger oxyhemoglobin. This fact suggests that NO+ ions rather than neutral NO molecules are responsible for the enzyme inactivation due to nitrosation of its structures. The enhancement of catalase inhibition in the presence of halide ions and thiocyanate might be caused by nitrosyl halide formation. The latter protected nitrosonium ions against hydrolysis, thereby ensuring their transfer to the targets in enzyme molecules. The addition of oxyhemoglobin plus iron chelator o-phenanthroline destroying DNIC sharply attenuated the inhibitory effect of DNIC on catalase. o-Phenanthroline added alone did not influence this effect. Oxyhemoglobin is suggested to scavenge nitrosonium ions released from decomposing DNIC, thereby preventing catalase nitrosation. The mixture of oxyhemoglobin and o-phenanthroline did not affect the inhibitory action of nitrite or S-nitrosothiols on catalase.
Keywords: dinitrosyl iron complex (DNIC); nitro compounds; nitroso compounds; catalase; inhibition

Isolation and characterization of extracellular α-galactosidases from Penicillium canescens by O. A. Sinitsyna; E. A. Fedorova; I. M. Vakar; E. G. Kondratieva; A. M. Rozhkova; L. M. Sokolova; T. M. Bubnova; O. N. Okunev; A. M. Chulkin; Y. P. Vinetsky; A. P. Sinitsyn (97-106).
Two α-galactosidases were purified to homogeneity from the enzymatic complex of the mycelial fungus Penicillium canescens using chromatography on different sorbents. Substrate specificity, pH-and temperature optima of activity, stability under different pH and temperature conditions, and the influence of effectors on the catalytic properties of both enzymes were investigated. Genes aglA and aglC encoding α-galactosidases from P. canescens were isolated, and amino acid sequences of the proteins were predicted. In vitro feed testing (with soybean meal and soybean byproducts enriched with galactooligosaccharides as substrates) demonstrated that both α-galactosidases from P. canescens could be successfully used as feed additives. α-Galactosidase A belonging to the 27th glycosyl hydrolase family hydrolyzed galactopolysaccharides (galactomannans) and α-galactosidase C belonging to the 36th glycosyl hydrolase family hydrolyzed galactooligosaccharides (stachyose, raffinose, etc.) of soybean with good efficiency, thus improving the digestibility of fodder.
Keywords: α-galactosidase; Penicillium canescens ; HPLC; stachyose; raffinose; galactomannan; feed additives; soybean

Spectroelectrochemistry was used to determine the midpoint redox potentials of heme cofactors of the caa 3-type cytochrome oxidase from the alkaliphilic bacterium Bacillus pseudofirmus FTU. The apparent midpoint potentials (E m app ) for the most prominent transitions of hemes a and a 3 (+193 and +334 mV, respectively) were found to be similar to the values reported for other enzymes with high homology to the caa 3-type oxidase. In contrast, the midpoint potential of the covalently bound cytochrome c (+89 mV) was 150–170 mV lower than in cytochromes c, either low molecular weight or covalently bound to the caa 3 complex in all known aerobic neutralophilic and thermo-neutralophilic bacteria. Such an unusually low redox potential of the covalently bound cytochrome c of the caa 3-type oxidase of alkaliphilic bacteria, together with high redox potentials of hemes a and a 3, ensures more than twice higher difference in redox potentials inside the respiratory complex compared to the homologous mitochondrial enzyme. The energy released during this redox transition might be stored in the transmembrane H+ gradient even under low Δp in the alkaline environment of the bacteria at the expense of a significant increase in ΔG of the coupled redox reaction.
Keywords: Bacillus pseudofirmus ; alkaliphile; cytochrome c ; caa 3 ; cytochrome oxidase; redox potential

The possible functions of a light-induced electron transfer to oxygen in the photosynthetic electron transport chain of higher plant chloroplasts are considered. The thermodynamic preconditions, as well as the experimental data about the participations of ferredoxin, the components of photosystems I and II, and plastoquinone in oxygen reduction are examined. It is concluded that, even in the presence of ferredoxin and ferredoxin + NADP+, oxygen reduction is carried out mainly by the membrane-bound carriers of the photosynthetic electron transport chain. The hypothesis is put forward that most superoxides, which are produced by reduction of O2 molecules by the intramembrane components of the acceptor side of photosystem I, are reduced within the membrane by the plastohydroquinone molecules to the hydrogen peroxide. It is assumed that the H2O2 molecules that originate as the result of this process serve for signaling about the redox state of the plastoquinone pool.
Keywords: chloroplast; thylakoid membrane; electron transport chain; plastoquinone pool; oxygen; superoxide radical