Papers by Tishkov Vladimir I.
FEBS Letters, 1999
NAD+‐dependent formate dehydrogenase (EC 1.2.1.2, FDH) from methylotrophic bacterium Pseudomonas ... more NAD+‐dependent formate dehydrogenase (EC 1.2.1.2, FDH) from methylotrophic bacterium Pseudomonas sp.101 exhibits the highest stability among the similar type enzymes studied. To obtain further increase in the thermal stability of FDH we used one of general approaches based on hydrophobization of protein α‐helices. Five serine residues in positions 131, 160, 168, 184 and 228 were selected for mutagenesis on the basis of (i) comparative studies of nine FDH amino acid sequences from different sources and (ii) with the analysis of the ternary structure of the enzyme from Pseudomonas sp.101. Residues Ser‐131 and Ser‐160 were replaced by Ala, Val and Leu. Residues Ser‐168, Ser‐184 and Ser‐228 were changed into Ala. Only Ser/Ala mutations in positions 131, 160, 184 and 228 resulted in an increase of the FDH stability. Mutant S168A was 1.7 times less stable than the wild‐type FDH. Double mutants S(131,160)A and S(184,228)A and the four‐point mutant S(131,160,184,228)A were also prepared and...
Acta Naturae, 2010
РеФеРАт Исследование кинетики инактивации пероксидом водорода мутантной формиатдегидрогеназы из б... more РеФеРАт Исследование кинетики инактивации пероксидом водорода мутантной формиатдегидрогеназы из бактерий Pseudomonas sp. 101 (PseFDH) с заменой Cys255Ala свидетельствует, что взаимодействие фермента с инактивирующим агентом протекает по простому бимолекулярному механизму. В присутствии избытка пероксида водорода потеря активности описывается кинетикой реакции первого порядка. Поэтому наблюдаемая эффективная константа скорости инактивации первого порядка, полученная для различных форм ФДГ при постоянной концентрации H 2 O 2 , может быть использована в качестве количественной характеристики стабильности этих форм. Показано, что два остатка цистеина, расположенные в формиат-и кофермент-связывающем доменах активного центра (Cys145 и Cys255 соответственно), при инактивации H 2 O 2 вносят одинаковый вклад в стабильность фермента, а остаток Cys354 не является существенным. Сравнение кинетики инактивации PseFDH дикого типа, мутанта PseFDH Cys145Ser/Cys255Ala и стресс-индуцируемых формиатдегидрогеназ из бактерий Staphylococcus aureus, растений Arabidopsis thaliana и сои Glycine max свидетельствует, что «стрессовые» ФДГ минимум в 20 раз более стабильны против инактивации пероксидом водородм, чем PseFDH, экспрессия которой индуцируется при росте бактерий Pseudomonas sp. 101 на метаноле, но не в условиях стрессовых воздействий. КлЮЧевые СловА формиатдегидрогеназа, пероксид водорода, инактивация, стресс, мутантный фермент.
Moscow University Chemistry Bulletin, 2014
Tobacco anionic peroxidase (TOP) mutant Ile37Met was produced by site directed mutagenesis to mim... more Tobacco anionic peroxidase (TOP) mutant Ile37Met was produced by site directed mutagenesis to mimic soybean peroxidase (SBP), in which Met37 is responsible for increased thermal stability. TOP Ile37Met was expressed in E. coli BL21(DE3) CodonPlus in the form of bodies. The expression level of the constructed enzyme was approximately 40% of the total E. coli protein. The enzyme was reactivated into an active and soluble form via a refolding procedure that was earlier developed for wild type TOP. The substrate specificity, catalytic activity, and thermal stability of TOP Ile37Met were investigated. It was shown that the introduction of the Ile37Met mutation does not increases the stability of the enzyme; on the contrary, it leads to a reduction in the catalytic properties of the enzyme.
FEBS Letters, 1996
Gln313 and His332 residues in the active centre of NAD+‐dependent formate dehydrogenase (EC 1.2.1... more Gln313 and His332 residues in the active centre of NAD+‐dependent formate dehydrogenase (EC 1.2.1.2, FDH) from the bacterium Pseudomonas sp. 101 are conserved in all FDHs and are equivalent to the glutamate‐histidine pair in active sites of d‐specific 2‐hydroxyacid dehydrogenases. Two mutants of formate dehydrogenase from Pseudomonas sp. 101, Gln313Glu and His332Phe, have been obtained and characterised. The Gln313Glu mutation shifts the pK of the group controlling formate binding from less than 5.5 in wild‐type enzyme to 7.6 thus indicating that Gln313 is essential for the broad pH affinity profile towards substrate. His332Phe mutation leads to a complete loss of enzyme activity. The His332Phe mutant is still able to bind coenzyme but not substrate or analogues. The role of histidine in the active centre of FDH is discussed. The protonation state of His332 is not critical for catalysis but vital for substrate binding. A partial positive charge on the histidine imidazole, required f...
Journal of Biotechnology, 2010
Moscow University Chemistry Bulletin
Acta Naturae
Formate dehydrogenase from Pseudomonas sp. 101 bacterium (PseFDH, EC 1.2.1.2) is a research model... more Formate dehydrogenase from Pseudomonas sp. 101 bacterium (PseFDH, EC 1.2.1.2) is a research model for the elucidation of the catalytic mechanism of 2-oxyacid D-specific dehydrogenases enzyme superfamily. The enzyme is actively used for regeneration of the reduced form of NAD(P)H in chiral synthesis with oxidoreductases. A multi-point mutant PseFDH SM4S with an improved thermal and chemical stability has been prepared earlier in this laboratory. To further improve the properties of the mutant, additional single-point replacements have been introduced to generate five new PseFDH mutants. All new enzymes have been highly purified, and their kinetic properties and thermal stability studied using analysis of thermal inactivation kinetics and differential scanning calorimetry. The E170D amino acid change in PseFDH SM4S shows an increase in thermal stability 1.76- and 10-fold compared to the starting mutant and the wild-type enzyme, respectively.
Bioluminescence and Chemiluminescence, 2007
Molecules, 2022
Ginsenoside Rh2 increases the efficacy of doxorubicin (DOX) treatment in murine models of solid a... more Ginsenoside Rh2 increases the efficacy of doxorubicin (DOX) treatment in murine models of solid and ascites Ehrlich’s adenocarcinoma. In a solid tumor model (treatment commencing 7 days after inoculation), DOX + Rh2 co-treatment was significantly more efficacious than DOX alone. If treatment was started 24 h after inoculation, the inhibition of tumor growth of a solid tumor for the DOX + Rh2 co-treatment group was complete. Furthermore, survival in the ascites model was dramatically higher for the DOX + Rh2 co-treatment group than for DOX alone. Mechanisms underlying the combined DOX and Rh2 effects were studied in primary Ehrlich’s adenocarcinoma-derived cells and healthy mice’s splenocytes. Despite the previously established Rh2 pro-oxidant activity, DOX + Rh2 co-treatment revealed no increase in ROS compared to DOX treatment alone. However, DOX + Rh2 treatment was more effective in suppressing Ehrlich adenocarcinoma cell adhesion than either treatment alone. We hypothesize that t...
Copyright © 2010 Park-media, Ltd. This is an open access article distributed under the Creative C... more Copyright © 2010 Park-media, Ltd. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT The kinetics of the thermal inactivation of recombinant wild-type formate dehydrogenase from Candida boidinii yeast was studied in the temperature range of 53–61oC and pH 6.0, 7.0, and 8.0. It was shown that the loss of the enzyme’s activity proceeds via a monomolecular mechanism. Activation parameters ΔН ≠ and ΔS ≠ were calculated based on the temperature relations dependence of inactivation rate constants according to the transition state theory. Both parameters are in a range that corresponds to globular protein denaturation processes. Optimal conditions for the stability of the enzyme were high concentrations of the phosphate buffer or of the enzyme substrate sodium formate at pH = 7.0.
Biotechnology and Applied Biochemistry, 1993
Using two degenerate 20‐ and 23‐mer oligonucleotide probes, the gene encoding NAD(+)‐dependent fo... more Using two degenerate 20‐ and 23‐mer oligonucleotide probes, the gene encoding NAD(+)‐dependent formate dehydrogenase (FDH) (EC 1.2.1.2) was shown to lie within a 3.5 kb PstI fragment of the chromosomal DNA of methylotrophic bacterium Pseudomonas sp. 101. A phasmid library was prepared in the lambda pSL5 vector with partial EcoRI‐digested DNA from Pseudomonas sp. 101. The 12 clones selected contained three types of phasmid: lambda pFDH1, lambda pFDH2 and lambda pFDH3 (with inserts of 15, 17.6 and 18.5 kb respectively). The inserts contained the same 15 kb EcoRI and 3.5 kb PstI fragments and included the complete FDH gene. Further subcloning of the insert resulted in plasmid pFDH2 and a 2.32 kb HindIII‐BgIII fragment. Active enzyme was expressed in Escherichia coli TG1 (pFDH2) strain under control of a lac promoter.
Moscow University Chemistry Bulletin, 2020
The 3D full-atom model of the whole-size CYP102A1 from Bacillus megaterium (cytochrome P450 BM3) ... more The 3D full-atom model of the whole-size CYP102A1 from Bacillus megaterium (cytochrome P450 BM3) has been constructed using molecular modeling methods. The structure model was constructed using crystal structures of the separate FAD-binding domain (PDB ID: 4DQK) and the complex of FMN-binding and monooxygenase domains (PDB ID: 1BVY). Modeling procedure included analysis of the domains’ surfaces to find the orientation with maximum inter-subunit contacts. The overall configuration of the obtained complex was optimized using molecular dynamics. The final full-atom structure model shows rather tight interactions between FAD- and FMN-binding domains due to 10 inter-domain hydrogen bonds and hydrophobic interactions between three pairs of amino acid residues. This 3D model can be used for structure-function studies and rational design of the enzyme as well as for construction of hybrid supramolecular structures of biocatalysts with cytochrome P450 BM3.
Moscow University Chemistry Bulletin, 2018
Background: L-carnosine can suppress increased renal sympathetic nerve activity (SNA) during the ... more Background: L-carnosine can suppress increased renal sympathetic nerve activity (SNA) during the renal ischemia by its action on the central nervous system and that this suppressive effect is probably responsible for the renoprotection against ischemic/reperfusion induced renal injury. In addition, the renoprotective effect of Lcarnosine on ischemic acute renal failure seems to be induced by its conversion to L-histidine and L-histamine, and it is mediated through the activation of histamine H3 receptors in the central nervous system. Objective: Studying the effect of carnosine as a potential antioxidant agent on cadmium-induced lipid peroxidation and renal oxidative stress in aged rats. Materials and Methods: The present study was performed on 45 aged female Wistar rats, weighing at the start of the study between 280-380 g. Animals were randomly divided into the following equal groups: Control group, Cadmium group, and Carnosine treated group. Blood samples were collected and were subjected to measurement of serum urea, creatinine, albumin, malondialdehyde (MDA), superoxide dismutase (SOD), nitric oxide (NO) levels, tumor necrosis factor-α (TNF-α), interleukin10 (IL-10) levels, renal tissue tumor necrosis factor-α (TNF-α), superoxide dismutase (SOD), nitric oxide (NO) levels and measurement of cadmium (Cd) level in blood and renal tissue. Also, histopathological study of rat kidneys was performed. Results: Significant increases in serum urea and creatinine, MDA, IL10, serum and renal tissue NO, TNF-α, blood and renal tissue cadmium levels were encountered in cadmium group compared to control group. Carnosine treatment significantly decreased serum urea, creatinine, MDA, IL10, serum and renal tissue NO, TNF-α, blood and renal tissue cadmium levels compared to cadmium group though the levels were still significantly higher than control group. Serum albumin, serum and renal tissue SOD levels significantly decreased in cadmium group compared to control group. By treatment with carnosine, significant increases were observed compared to cadmium group though still significantly less compared to control group. Conclusion: Increased lipid peroxides induced by cadmium toxicity in aged rats may implicate the renal oxidative stress. Moreover, pretreatment with carnosine successively boosted the antioxidant system through several mechanisms such as scavenging/neutralizing free radicals, regulating enzymatic/non enzymatic antioxidants. However, future work is needed to confirm our results.
Acta Naturae, 2015
It has been shown by an X-ray structural analysis that the amino acid residues Ala198, which are ... more It has been shown by an X-ray structural analysis that the amino acid residues Ala198, which are located in the coenzyme-binding domain of NAD+-dependent formate dehydrogenases (EC 1.2.1.2., FDH) from bacteria Pseudomonas sp.101 and Moraxella sp. C-1 (PseFDH and MorFDH, respectively), have non-optimal values of the angles and . These residues were replaced with Gly by site-directed mutagenesis. The mutants PseFDH A198G and MorFDH A198G were expressed in E.coli cells and obtained in active and soluble forms with more than 95% purity. The study of thermal inactivation kinetics showed that the mutation A198G results in a 2.5- fold increase in stability compared to one for the wild-type enzymes. Kinetic experiments indicate that A198G replacement reduces the K M NAD+ value from 60 to 35 and from 80 to 45 M for PseFDH and MorFDH, respectively, while the K M HCOO- value remains practically unchanged. Amino acid replacement A198G was also added to the mutant PseFDH D221S with the coenzyme ...
Moscow University Chemistry Bulletin, 2018
Activation of antihypoxic program under the action of a number of transition and heavy metals has... more Activation of antihypoxic program under the action of a number of transition and heavy metals has been studied using cell-based HIF1 ODD-luc and HRE-luc reporters. It has been demonstrated that Au3+, Pb2+, Sn2+, Hg2+ are weak HIF1 ODD-luc activators, likely reflecting their weak competition for the ironbinding site in the active center of HIF prolyl hydroxylase. Metals capable of replacing iron–Mn2+, Zn2+, Cu2+ и Ni2+–activate at high submillimolar concentrations, which indicates low permeability of the cell membrane for transition metals. The highest activation is observed for Co2+ and Cd2+, however, Cd2+ is highly toxic even at 10 μM, in contrast to Co2+, which activates both reporters without toxicity signs up to 25 μM for 24 h. A significant activation by Co2+ is observed already in low micromolar range of concentrations, which can be recommended for use in hypoxia mimicking.
Acta Naturae, 2017
The bacteriolytic activity of interleukin-2 and chicken egg lysozyme in the presence of various s... more The bacteriolytic activity of interleukin-2 and chicken egg lysozyme in the presence of various substances has been studied. Glycine and lysine do not affect the activity of interleukin-2 but increase that of lysozyme, showing a bell-shape concentration dependence peaking at 1.5 mM glycine and 18 mM lysine. Arginine and glutamate activate both interleukin-2 and lysozyme with a concentration dependence of the saturation type. Aromatic amino acids have almost no effect on the activity of both interleukin-2 and lysozyme. Aromatic amines, tryptamine, and tyramine activate interleukin-2 but inhibit lysozyme. Peptide antibiotics affect interleukin and lysozyme similarly and exhibit maximum activity in the micromolar range of antibiotics. Taurine has no effect on the activity of interleukin-2 and lysozyme. Mildronate showed no influence on lysozyme, but it activated interleukin-2 with the activity maximum at 3 mM. EDTA activates both interleukin-2 and lysozyme at concentrations above 0.15 mM.
Russian Chemical Bulletin, 2018
Reporters expressing fusion proteins of HIF2 and HIF3 C-terminal oxygen degradable domain (ODD) w... more Reporters expressing fusion proteins of HIF2 and HIF3 C-terminal oxygen degradable domain (ODD) with the firefly luciferase, HIF2 ODD-luc and HIF3 ODD-luc, were constructed and briefly characterized. Stable neuroblastoma cell lines expressing either reporter were generated, and their response to the known HIF prolyl hydroxylase inhibitors: dimethyloxalylglycine, ciclopirox, and adaptaquin, was studied and compared with the HIF1 ODD-luc reporter. The HIF2 ODD-luc reporter exhibited the highest sensitivity: its response in absolute luminescence value was almost an order of magnitude higher than that of the HIF1 ODD-luc reporter. The new reporter can be used for a fine discrimination of enzyme inhibitors stabilizing HIF2, and further structural optimization of adaptaquin discovered earlier by using the HIF1 ODD-luc reporter. The higher sensitivity of HIF2 ODD-luc reporter could be most likely explained by the lower affinity of the endogenous enzyme for this HIF isoform in comparison with the two others, which also resulted in the increased efficiency of inhibitors under the reporter assay conditions.
Moscow University Chemistry Bulletin, 2018
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Papers by Tishkov Vladimir I.