Biochemistry (Moscow) (v.74, #8)

Autonomous 3′→5′ exonucleases (AE) are not bound covalently to DNA polymerases, but they are often included into the replicative complexes. Intracellular AE overproduction in bacteria results in sharp suppression of mutagenesis, whereas inactivation of these enzymes in bacteria and fungi leads to an increase in mutagenesis frequency by 2–3 orders of magnitude. Correction of DNA polymerase errors in vitro occurs after addition of AE to the incubation medium. This correction is clearly manifested under conditions of mutational stress (during induced but not spontaneous mutagenesis), for instance, with an imbalance of dNTPs — error-prone conditions. At equimolar dNTP (error-free conditions), the correction is relatively weak. The gene knockout of both alleles of the major AE gene in mice does not influence spontaneous mutagenesis though a substantial increase could be expected. The frequency of induced mutagenesis has not been yet measured, though the inactivation of AE could increase the frequency of mutagenesis. Complete inactivation of the major AE leads to inflammatory myocarditis and a 5-fold reduction of life span of mice. Dominant heterozygous mutations were found in various loci of the AE gene, which caused the development of Aicardi-Goutieres (autosomal recessive encephalopathy) syndrome, familial chilblain lupus, systemic lupus erythematosus, retinal vasculopathy, and cerebral leukodystrophy. In the nucleus, AE have a corrective function, but after transition into cytoplasm these enzymes destroy aberrant DNA that appears during replication and thereby save the cells from autoimmune diseases. Depending on their intracellular localization, AE carry out various biological functions but employ the same mechanism of the catalyzed reactions.
Keywords: correction of DNA polymerase errors; mutant forms of proteins; autoimmune inflammations

Effect of reactive center loop structure of antichymotrypsin on inhibition of duodenase activity by T. S. Zamolodchikova; N. A. Popykina; I. P. Gladysheva; N. I. Larionova (824-833).
Interaction between duodenase (a granase family member) from bovine duodenal mucosa and recombinant antichymotrypsin (rACT) and its P1 variants has been studied. Association rate constants (k a) were 11, 6.8, and 17 mM−1·sec−1 for rACT, ACT L358M, and ACT L358R, respectively. Natural antitrypsin (AT) compared to ACT was a 20 times more effective duodenase inhibitor (in terms of k a). Duodenase interacted with P1 variants of ACT via a suicide mechanism with stoichiometry of the process SI = 1.2. The nature of the P1 residue of the inhibitor did not influence the interaction if other residues did not meet conformational requirements of the duodenase substrate-binding pocket. Also, interaction of duodenase with ACT variants containing residues from AT reaction center loop (rACT P2-P3′, rACT P3-P4′, rACT P4-P3′, and rACT P6-P4′) was studied. The inhibition type ([E]0 = 1·10−7 M, 25°C) was revealed to be reversible-like, and efficacy of inhibition decreased with increase in the substituted part of the reactive center loop. Constants of inhibition (K i) were measured. Efficacy of interaction between the enzyme (duodenase) and inhibitor depends on topochemical correspondence between a substrate-binding pocket of the enzyme and substrate structure.
Keywords: duodenase; serpins; conformational specificity; granase

RNA-dependent RNA polymerase of hepatitis C virus: Study on inhibition by α,γ-diketo acid derivatives by M. V. Kozlov; K. M. Polyakov; S. E. Filippova; V. V. Evstifeev; G. S. Lyudva; S. N. Kochetkov (834-841).
It is supposed that α,γ-diketo acids (DKAs) inhibit the activity of hepatitis C virus RNA-dependent RNA poly-merase (RdRP HCV) via chelation of catalytic magnesium ions in the active center of the enzyme. However, DKAs display noncompetitive mode of inhibition with respect to NTP substrate, which contradicts the proposed mechanism. We have examined the NTP substrate entry channel and the active site of RdRP HCV for their possible interaction with DKAs. The substitutions R48A, K51A, and R222A greatly facilitated RdRP inhibition by DKAs and simultaneously increased K m values for UTP substrate. Interestingly, C223A was the only one of a number of substitutions that decreased K m(UTP) but facilitated the inhibitory action of DKAs. The findings allowed us to model an enzyme-inhibitor complex. According to the proposed model, DKAs introduce an additional Mg2+ ion into the active site of the enzyme at a stage of phosphodiester bond formation, which results in displacement of the NTP substrate triphosphate moiety to a catalytically inactive binding mode. This mechanism, in contrast to the currently adopted one, explains the noncompetitive mode of inhibition.
Keywords: hepatitis C virus; RNA-dependent RNA polymerase; α,γ-diketo acid derivatives; inhibition mechanism; molecular modeling

Finding of endopolyphosphatase activity in the yeast Saccharomyces cerevisiae by L. P. Lichko; T. V. Kulakovskaya; E. V. Kulakovskaya; I. S. Kulaev (842-845).
Endopolyphosphatase activity has been revealed in cytosol preparations of the yeast Saccharomyces cerevisiae with inactivated PPX1 and PPN1 genes encoding exopolyphosphatases. The enzyme cleaves inorganic polyphosphates with chain length of 15 to 208 phosphate residues to shorter chains without the release of orthophosphate (Pi). The long chain polyphosphates are cleaved with preference over the short ones. Heparin, a known inhibitor of exopolyphosphatases, represses this activity. The endopolyphosphatase activity is not stimulated by Mg2+ or Co2+, in contrast to exopolyphosphatases. This activity along with a pyrophosphatase is supposed to be responsible for polyphosphate utilization as a phosphate reserve in a mutant devoid of exopolyphosphatases.
Keywords: inorganic polyphosphates; endopolyphosphatase; exopolyphosphatase; cytosol; PPX1 and PPN1 mutant; Saccharomyces cerevisiae

Femtosecond phase of charge separation in reaction centers of Chloroflexus aurantiacus by A. G. Yakovlev; T. A. Shkuropatova; L. G. Vasilieva; A. Ya. Shkuropatov; V. A. Shuvalov (846-854).
Difference absorption spectroscopy with temporal resolution of ∼20 fsec was used to study the primary phase of charge separation in isolated reaction centers (RCs) of Chloroflexus aurantiacus at 90 K. An ensemble of difference (light-minus-dark) absorption spectra in the 730–795 nm region measured at −0.1 to 4 psec delays relative to the excitation pulse was analyzed. Comparison with analogous data for RCs of HM182L mutant of Rhodobacter sphaeroides having the same pigment composition identified the 785 nm absorption band as the band of bacteriopheophytin ΦB in the B-branch. By study the bleaching of this absorption band due to formation of Φ B , it was found that a coherent electron transfer from P* to the B-branch occurs with a very small delay of 10–20 fsec after excitation of dimer bacteriochlorophyll P. Only at 120 fsec delay electron transfer from P* to the A-branch occurs with the formation of bacteriochlorophyll anion B A absorption band at 1028 nm and the appearance of P* stimulated emission at 940 nm, as also occurs in native RCs of Rb. sphaeroides. It is concluded that a nuclear wave packet motion on the potential energy surface of P* after a 20-fsec light pulse excitation leads to the coherent formation of the P+Φ B and P+B A states.
Keywords: photosynthesis; charge separation; reaction center; wave packet; electron transfer

Specificity of oxidation of linoleic acid homologs by plant lipoxygenases by I. R. Chechetkin; E. V. Osipova; N. B. Tarasova; F. K. Mukhitova; M. Hamberg; Y. V. Gogolev; A. N. Grechkin (855-861).
The lipoxygenase-catalyzed oxidation of linoleic acid homologs was studied. While the linoleic acid oxidation by maize 9-lipoxygenase (9-LO) specifically produced (9S)-hydroperoxide, the dioxygenation of (11Z,14Z)-eicosadienoic (20:2) and (13Z,16Z)-docosadienoic (22:2) acids by the same enzyme lacked regio- and stereospecificity. The oxidation of 20:2 and 22:2 by 9-LO afforded low yields of racemic 11-, 12-, 14-, and 15-hydroperoxides or 13- and 17-hydroperoxides, respectively. Soybean 13-lipoxygenase-1 (13-LO) specifically oxidized 20:2, 22:2, and linoleate into (ω6S)-hydroperoxides. Dioxygenation of (9Z,12Z)-hexadecadienoic acid (16:2) by both 9-LO and 13-LO occurred specifically, affording (9S)- and (13S)-hydroperoxides, respectively. The data are consistent with the “pocket theory of lipoxygenase catalysis” (i.e. with the penetration of a substrate into the active center with the methyl end first). Our findings also demonstrate that the distance between carboxyl group and double bonds substantially determines the positioning of substrates within the active site.
Keywords: lipoxygenase; enzyme-substrate interaction; polyenoic fatty acid; oxylipin

In the present paper we describe studies on molecular mechanisms of protein-protein interactions between cytochrome P450 3A4 (CYP3A4) and cytochrome b 5, the latter being incorporated into the artificial recombinant protein Hmwb 5-EGFP containing full-length cytochrome b 5 (functional module) and a mutant form of the green fluorescent protein EGFP (signal module) fused into a single polypeptide chain. It is shown that cytochrome b 5 within the fusion protein Hmwb 5-EGFP can be reduced by NADPH-cytochrome P450 reductase in the presence of NADPH, the rate of reduction being dependent on solution ionic strength, indicating that the signal module does not prevent the interaction of the flavo- and hemeproteins. Interaction of cytochrome P450 3A4 and Hmwb 5-EGFP was estimated based on spin equilibrium shift of cytochrome P450 3A4 to high-spin state in the presence of Hmwb 5-EGFP, as well as based on steady-state fluorescence anisotropy of the EGFP component of the fusion protein in the presence of CYP3A4. The engineering of chimeric protein Hmwb 5-EGFP gives an independent method to determine dissociation constant for the complex of cytochrome P450 and cytochrome b 5 that is less sensitive to environmental factors compared to spectrophotometric titration used before. Reconstitution of catalytic activity of cytochrome P450 3A4 in the reaction of testosterone 6β-hydroxylation in the presence of Hmwb 5-EGFP indicates that cytochrome b 5 in the fusion protein is able to stimulate the hydroxylation reaction. Using other fusion proteins containing either cytochrome b 5 or its hydrophilic domain to reconstitute catalytic activity of cytochrome P450 3A4 showed that the hydrophobic domain of cytochrome b 5 participates not only in hemeprotein interaction, but also in electron transfer from cytochrome b 5 to cytochrome P450.
Keywords: microsomal cytochrome b 5 ; green fluorescent protein; cytochrome P450 3A4; testosterone 6β-hydroxylation

Studies on functional role of DNA methylation within the FXYD5-COX7A1 region of human chromosome 19 by Y. V. Skvortsova; T. L. Azhikina; E. A. Stukacheva; E. D. Sverdlov (874-881).
We used the Rapid Identification of Genomic Splits technique to get a detailed methylation landscape of a 1-megabase-long human genome region (FXYD5-COX7A1, chromosome 19) in normal and tumor lung tissues and in the A549 lung cancer cell line. All three samples were characterized by an essentially uneven density of unmethylated sites along the fragment. Strikingly enough, the distribution of hypomethylated regions did not correlate with gene locations within the fragment. We also demonstrated that the methylation pattern of this long genomic DNA fragment was rather stable and practically unchanged in human lung cancer tissue as compared with its normal counterpart. On the other hand, the methylation landscape obtained for the A549 cell line (human lung carcinoma) in the USF2-MAG locus showed clear differences from that of the tissues mentioned above. A comparative analysis of transcriptional activity of the genes in this region demonstrated the general absence of direct correlation between methylation and expression, although some data suggest a possible role of methylation in the regulation of MAG expression through cis-regulatory elements. In total, our data provide new evidence for the necessity of revising currently prevailing views on the functional significance of methyl groups in genomic DNA.
Keywords: methylation profile; regulation of gene expression; MAG ; HAMP

Regulatory activity of heterologous gene-activator xlnR of Aspergillus niger in Penicillium canescens by Y. P. Vinetsky; A. M. Rozhkova; A. M. Chulkin; A. D. Satrutdinov; O. A. Sinitsyna; E. A. Fedorova; A. O. Bekkarevich; O. N. Okunev; A. P. Sinitsyn (882-887).
The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.
Keywords: xylanolytic activator; xlnR ; Penicillium canescens axhA promoter; Aspergillus niger

Prediction of amino acid residues protected from hydrogen-deuterium exchange in a protein chain by N. V. Dovidchenko; M. Yu. Lobanov; S. O. Garbuzynskiy; O. V. Galzitskaya (888-897).
We have investigated the possibility to predict protection of amino acid residues from hydrogen-deuterium exchange. A database containing experimental hydrogen-deuterium exchange data for 14 proteins for which these data are known has been compiled. Different structural parameters related to flexibility of amino acid residues and their amide groups have been analyzed to answer the question whether these parameters can be used for predicting the protection of amino acid residues from hydrogen-deuterium exchange. A method for prediction of protection of amino acid residues, which uses only the amino acid sequence of a protein, has been elaborated.
Keywords: hydrogen-deuterium exchange; secondary structure; hydrogen bond; B-factor; regions with irregular secondary structure

The Xenopus laevis 1-Cys-peroxiredoxin (peroxiredoxin 6, Prx6) gene was cloned and expressed in Escherichia coli. The enzymatic properties of the recombinant protein were characterized and compared to those of human Prx6. Xenopus laevis Prx6 has 224 amino acid residues including five Cys, one of which, Cys47, is located in the active center determining peroxidase activity. The stability and activity of X. laevis Prx6 relative to hydrogen peroxide and tret-butyl hydroperoxide are very similar to corresponding values for human Prx6. Both enzymes have temperature optimum at 37°C, but the clawed frog enzyme retains no less than 50% of activity over a wider temperature interval (10–50°C) than the human one (25–50°C). The expression of X. laevis prx6 at different stages of development was investigated. The level of gene expression increased during development, especially at stages 33–43 during formation of the lungs, when heartbeat and hatching begins.
Keywords: peroxiredoxin 6; thermostability; developmental stages; Xenopus laevis

Drought-induced changes in photosynthetic membranes of two wheat (Triticum aestivum L.) cultivars by I. M. Huseynova; S. Y. Suleymanov; S. M. Rustamova; J. A. Aliyev (903-909).
Two wheat (Triticum aestivum L.) cultivars contrasting in architectonics and differing in drought resistance, Azamatli-95 (short stature, vertically oriented small leaves, drought-tolerant) and Giymatli-2/17 (short stature, broad and drooping leaves, drought-sensitive), were studied. It was found that the content of CP I (115 kDa) and 63-kDa apoprotein P700 and also LHC II polypeptides increases slightly in the drought-resistant cv. Azamatli-95 under extreme water supply limitation, while their content decreases in drought-sensitive cv. Giymatli-2/17. The intensity of synthesis of α- and β-subunits of CF1 (55 and 53.5 kDa) and 33–30.5 kDa proteins also decreases in the sensitive genotype. The intensity of short wavelength peaks at 687 and 695 nm sharply increases in the fluorescence spectra (77K) of chloroplasts from Giymatli-2/17 under water deficiency, and there is a stimulation of the ratio of fluorescence band intensity F687/F740. After exposure to drought, cv. Giymatli-2/17 shows a larger reduction in the actual PS II photochemical efficiency of chloroplasts than cv. Azamatli-95.
Keywords: wheat genotypes; photosynthetic membrane; proteins; fluorescence; photosystems; water stress

Binding of ATP and its derivatives to selenophosphate synthetase from Escherichia coli by Y. V. Preabrazhenskaya; I. Y. Kim; T. C. Stadtman (910-916).
Mechanistically similar selenophosphate synthetases (SPS) have been isolated from different organisms. SPS from Escherichia coli is an ATP-dependent enzyme with a C-terminal glycine-rich Walker sequence that has been assumed to take part in the first step of ATP binding. Three C-terminally truncated mutants of SPS, containing the N-terminal 238 (SPS238), 262 (SPS262), and 332 (SPS332) amino acids of the 348-amino-acid protein, have been extracted from cell pellets, and two of these (SPS262 and SPS332) have been purified to homogeneity. SPS238 has been obtained in a highly purified form. Binding of the fluorescent ATP-derivative TNP-ATP and Mn-ATP to the proteins was examined for all truncated mutants of SPS and a catalytically inactive C17S mutant. It has been shown that TNP-ATP can be used as a structural probe for ATP-binding sites of SPS. We observed two TNP-ATP binding sites per molecule of enzyme for wild-type SPS and SPS332 mutant and one TNP-ATP binding site for SPS238 mutant. The stoichiometry of Mn-ATP-binding was 2 mol of ATP per mol of protein determined with [14C]ATP by HPLC gel-filtration column chromatography under saturating conditions. The binding stoichiometries for SPS332, SPS262, and SPS238 were 2, 1.6, and 1, respectively. The C17S mutant exhibits about one third of wild type SPS TNP-ATP-binding ability and converts 12% of ATP in the ATPase reaction to ADP in the absence of selenide. The C-terminus contributes two thirds to the TNP-ATP binding; SPS238 likely has one ATP-binding site removed by truncation.
Keywords: selenophosphate synthetase; truncated mutants; ATP-binding; fluorescence enhancement

Expression of rol agrobacterial oncogenes in plant cell cultures is known to result in activation of secondary metabolite biosynthesis. In the present work, we show that rolC can activate expression of key genes of secondary metabolism using the rolC-transgenic culture of Eritrichium sericeum producing caffeic acid metabolites (rosmarinic acid and rabdosiin) as an example. Increased content of rosmarinic acid in the rolC-transformed callus culture resulted from transcriptional activation of members of the CYP98 family of plant cytochrome P450-containing monooxygenase genes. The rolC gene expression led to increased transcript abundance of the CYP98A3 subfamily members, which are closely related homologs of CYP98A6 of Lithospermum erythrorhizon and are known to be key genes in rosmarinic acid biosynthesis. In contrast, expression of other CYP genes, such as CYP98A1 and CYP98A2, which are not implicated in rosmarinic acid biosynthesis, was not activated in the rolC-transformed calluses. These results are indicative of selective effect of rolC on transcription of particular genes implicated in secondary metabolism.
Keywords: rolC oncogene; cytochrome P450 monooxygenases; CYP98

Molecular mechanisms of imidazole and benzene ring binding in proteins by A. V. Zhuravlev; B. F. Shchegolev; E. V. Savvateeva-Popova; A. V. Popov (925-932).
Aromatic bonds of amino acid radicals play an important role in arrangement of protein primary structure. Previously, the existence of a number of preferable conformations of aromatic dimers was shown theoretically and experimentally, the best known of which are parallel-displaced and perpendicular T conformations. To reveal principles that define preference of various conformations for His-His and Phe-His dimers, non-empirical quantum-chemical calculations of diimidazole and benzene-imidazole were carried out. Calculations were performed using the 6-31G** basis with account for electronic correlations in frames of MP2 and MP4 methods of perturbation theory. Comparative analysis of energetic and geometric parameters of the systems points to the preference of stacking contact or classical hydrogen bond in diimidazole. On the contrary, T conformation is maximally advantageous for benzene-imidazole.
Keywords: stacking interaction; hydrogen bond; diimidazole; benzene-imidazole

Nonaqueous titration of amino groups in polymeric matrix of plant cell walls by N. R. Meychik; Yu. I. Nikolaeva; I. P. Ermakov (933-937).
Nonaqueous titration was used for detection of free amino groups in the polymeric matrix of plant cell walls. The content of amino groups varied in the range 0.54–0.91 and total nitrogen in the range 1.0–4.2 mmol per gram dry mass of cell walls depending on the plant species. However, these data on the high content of free amino groups do not correlate with the present day concept that the nitrogen fraction in charged amino groups in plant cell wall proteins, which are assumed to be mainly amino groups of lysine and arginine residues, is about 10%. It is supposed that most detected free amino groups belong to the hydroxy-amino acids hydroxyproline and tyrosine that can be bound at the hydroxyl group with the carbohydrate part of glycoprotein or another structural cell wall polymer.
Keywords: amino groups; cell wall; plant roots