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2. On the roles of calcium and zinc ions in the formation of a catalytically active form of the metalloenzyme, l-alanyl-d-glutamate peptidase of the bacteriophage T5 (EndoT5)

Author

Dmitry A. Prokhorov, Galina V. Mikoulinskaia, Alexander Y. Yegorov, Vladimir N. Uversky, Victor P. Kutyshenko, and Nikolai V. Molochkov

Subjects
Models, Molecular, Circular dichroism, Magnetic Resonance Spectroscopy, Protein Conformation, Stereochemistry, Metal ions in aqueous solution, 02 engineering and technology, Biochemistry, Active center, Viral Proteins, 03 medical and health sciences, Structural Biology, Bacteriophage T7, Catalytic Domain, Endopeptidases, Molecular Biology, Bacteriophage T5, Histidine, 030304 developmental biology, Ions, chemistry.chemical_classification, 0303 health sciences, biology, Chemistry, General Medicine, 021001 nanoscience & nanotechnology, biology.organism_classification, Tautomer, Enzyme Activation, NMR spectra database, Zinc, Enzyme, Amino Acid Substitution, Calcium, 0210 nano-technology, Protein Binding
Abstract

Structural consequences of the binding of metal ions (regulatory Ca2+ and catalytic Zn2+) to the metalloenzyme l-alanyl-d-glutamate peptidase of the bacteriophage T5 (Endo T5) and some of its analogues containing single amino acid substitutions in the active center were analyzed by nuclear magnetic resonance (NMR), circular dichroism (CD) and calorimetry. Analyses revealed that the native EndoT5 undergoes strong structural rearrangements as a result of Zn2+ binding. This structural rearrangement resulting in the formation of an active enzyme is completed by the Ca2+ binding. In this case, the NMR spectra uncover the tautomerism of the NH protons of histidine imidazoles responsible for the Zn2+ coordination. For the EndoT5 analogues with point substitutions in the Ca2+-binding site, similar conformational rearrangements are observed upon Zn2+ binding. However, no characteristic changes in the NMR spectra associated with the Ca2+ binding were detected. The roles of the proton exchange in the process of Ca2+-induced activation of the enzymatic activity of EndoT5 is discussed.

Published
2020
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3. Lysis of cells of diverse bacteria by <scp>l,d</scp> ‐peptidases of Escherichia coli bacteriophages RB43, RB49 and T5

Author

L.V. Dorofeeva, V.S. Shadrin, Sergei V. Chernyshov, Galina V. Mikoulinskaia, and A.V. Machulin

Subjects
Lysis, Lysin, Peptidoglycan, medicine.disease_cause, Coliphages, Applied Microbiology and Biotechnology, Bacterial cell structure, Microbiology, Bacteriophage, 03 medical and health sciences, chemistry.chemical_compound, Bacteriolysis, Cell Wall, Endopeptidases, medicine, Bacteriophage T5, Escherichia coli, 030304 developmental biology, 0303 health sciences, Bacteria, biology, 030306 microbiology, General Medicine, biology.organism_classification, Anti-Bacterial Agents, chemistry, Biotechnology
Abstract

Aims The objective of this work was to study the antibacterial specificity and antibacterial effect of endolysins isolated from colibacteriophages RB43, RB49 and T5-as manifested on the exponential and stationary cell cultures of diverse bacteria depending on the growth stage, structure of peptidoglycan (PG) and antibiotic resistance. Methods and results Enzyme activity was assayed by the spectrophotometric method. Antimicrobial activity was estimated by the number of colony forming units (CFUs), with the results represented as logarithmic units. Morphological examination of bacterial cells was conducted using phase-contrast and scanning electron microscopy. The enzymes EndoT5, endolysin of bacteriophage T5, EndoRB43, endolysin of bacteriophage RB43 and EndoRB49, endolysin of bacteriophage RB49 turned out to be much less bacteriospecific than the corresponding Escherichia coli phages; they lysed bacteria of the genera Bacillus, Cellulomonas and Sporosarcina, whose PGs had different structures (A1γ, A4α and A4β) and chemical modifications (amidation). The specific lytic activity of phage enzymes was independent of the antibiotic resistance of bacterial cells and was higher when the cells were in the exponential, rather than stationary, growth phase. The analysis of morphological changes showed that the intermediate stage of the endolysin-induced lysis of bacterial cells was the formation of spheroplasts and protoplasts. Conclusions Endolysins of colibacteriophages RB49, RB43 and T5 have a wide spectrum of antibacterial action, which includes a number of diverse micro-organisms with different PG structures. Significance and impact of the study This is a study of the bacterial selectivity of enzymes degrading bacterial cell wall in relation to the chemical structure of PG. It is shown that endolysins of bacteriophages RB49 and RB43 efficiently lyse cell wall of Gram-positive bacteria of the genus Bacillus and Gram-negative bacteria of the genus Pseudomonas (including an antibiotic-resistant strain). The number of bacterial cells is reduced by 3-6 orders of magnitude, which indicates good prospects for using these enzymes in biotechnology.

Published
2020
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4. Recent Origin of the Methacrylate Redox System in Geobacter sulfurreducens AM-1 through Horizontal Gene Transfer.

Author

Oksana V Arkhipova, Margarita V Meer, Galina V Mikoulinskaia, Marina V Zakharova, Alexander S Galushko, Vasilii K Akimenko, and Fyodor A Kondrashov

Subjects
Medicine, Science
Abstract

The origin and evolution of novel biochemical functions remains one of the key questions in molecular evolution. We study recently emerged methacrylate reductase function that is thought to have emerged in the last century and reported in Geobacter sulfurreducens strain AM-1. We report the sequence and study the evolution of the operon coding for the flavin-containing methacrylate reductase (Mrd) and tetraheme cytochrome с (Mcc) in the genome of G. sulfurreducens AM-1. Different types of signal peptides in functionally interlinked proteins Mrd and Mcc suggest a possible complex mechanism of biogenesis for chromoproteids of the methacrylate redox system. The homologs of the Mrd and Mcc sequence found in δ-Proteobacteria and Deferribacteres are also organized into an operon and their phylogenetic distribution suggested that these two genes tend to be horizontally transferred together. Specifically, the mrd and mcc genes from G. sulfurreducens AM-1 are not monophyletic with any of the homologs found in other Geobacter genomes. The acquisition of methacrylate reductase function by G. sulfurreducens AM-1 appears linked to a horizontal gene transfer event. However, the new function of the products of mrd and mcc may have evolved either prior or subsequent to their acquisition by G. sulfurreducens AM-1.

Published
2015
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5. Investigation of the calcium-induced activation of the bacteriophage T5 peptidoglycan hydrolase promoting host cell lysis

Author

Sergei V. Chernyshov, Dmitry A. Prokhorov, Irina V. Odinokova, Nikolai V. Molochkov, Galina V. Mikoulinskaia, Angelina O Kovalenko, and Victor P. Kutyshenko

Subjects
Models, Molecular, 0301 basic medicine, Circular dichroism, Lysis, Biophysics, chemistry.chemical_element, Calcium, Biochemistry, Bacterial cell structure, Biomaterials, Viral Proteins, 03 medical and health sciences, Escherichia coli, Bacteriophage T5, 030102 biochemistry & molecular biology, biology, Chemistry, Mutagenesis, Metals and Alloys, N-Acetylmuramoyl-L-alanine Amidase, Periplasmic space, biology.organism_classification, Enzyme Activation, 030104 developmental biology, Chemistry (miscellaneous), Cytoplasm, T-Phages
Abstract

Peptidoglycan hydrolase of bacteriophage T5 (EndoT5) is a Ca2+-dependent l-alanyl-d-glutamate peptidase, although the mode of Ca2+ binding and its physiological significance remain obscure. Site-directed mutagenesis was used to elucidate the role of the polar amino acids of the mobile loop of EndoT5 (111–130) in Ca2+ binding. The mutant proteins were purified to electrophoretic homogeneity, the overall structures were characterized by circular dichroism, and the calcium dissociation constants were determined via NMR spectroscopy. The data suggest that polar amino acids D113, N115, and S117 of EndoT5 are involved in the coordination of calcium ions by forming the core of the EF-like Ca2+-binding loop while the charged residues D122 and E123 of EndoT5 contribute to maintaining the loop net charge density. The results suggest that Ca2+ binding to the EndoT5 molecule could be essential for the stabilization of the long mobile loop in the catalytically active “open” conformation. The possible mechanism of Ca2+ regulation of EndoT5 activity during bacteriophage T5’s life cycle through the Ca2+ concentration difference between the cytoplasm and the periplasm of the host bacteria cell has been discussed. The study reveals valuable insight into the role of calcium in the regulation of phage-induced bacterial lysis.

Published
2019
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6. Methacrylate-Reducing Activity of Anaerobic Bacteria Anaeromyxobacter dehalogenans and Denitrovibrio acetiphilus

Author

E. N. Biryukova, V. V. Ashin, T. N. Abashina, G. V. Khokhlova, Galina V. Mikoulinskaia, and O. V. Arkhipova

Subjects
0303 health sciences, biology, 030306 microbiology, Chemistry, Periplasmic space, Reductase, Nitrate reductase, biology.organism_classification, Applied Microbiology and Biotechnology, Microbiology, 03 medical and health sciences, Biochemistry, Anaerobic bacteria, Gene, Intracellular, Bacteria, 030304 developmental biology, Anaeromyxobacter dehalogenans
Abstract

Periplasmic methacrylate-reducing activity was shown for anaerobic acetate-oxidizing gram-negative bacteria Anaeromyxobacter dehalogenans 2CP-1Т (class Deltaproteobacteria) and Denitrovibrio acetiphilus DSM 12809Т (class Deferribacteres), both possessing homologous genes for methacrylate redox system components. No acrylate reductase activity was revealed in these bacteria. A. dehalogenans and D. acetiphilus were also found to reduce fumarate, exhibiting periplasmic and intracellular fumarate reductase activity, respectively. D. acetiphilus was found to possess nitrate reductase activity of periplasmic and intracellular localization. The possible correspondence between reductase activities and the hypothetical proteins genes in the known genomes of A. dehalogenans and D. acetiphilus are discussed.

Published
2019
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7. Chemical synthesis of peptidoglycan mimetic–disaccharide-tetrapeptide conjugate and its hydrolysis by bacteriophage T5, RB43 and RB49 L-alanyl-D-glutamate peptidases

Author

Maxim Molchanov, Alexey Chulin, Viatcheslav N. Azev, Galina V. Mikoulinskaia, V. P. Kutyshenko, Dmitry A. Prokhorov, and Vladimir N. Uversky

Subjects
Peptidoglycan mimetic, Stereochemistry, Endolysin, Lysin, Biophysics, Biochemistry, Microbiology, General Biochemistry, Genetics and Molecular Biology, Bacterial cell structure, 03 medical and health sciences, Hydrolysis, chemistry.chemical_compound, NMR spectroscopy, Substrate hydrolysis, Chemical synthesis, Bacteriophage, Bacteriophage T5, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, biology, 030306 microbiology, EndoRB49, General Neuroscience, General Medicine, biology.organism_classification, EndoT5, Glycopeptide, Enzyme, Disaccharide-tetrapeptide conjugate, chemistry, Medicine, Peptidoglycan, EndoRB43, General Agricultural and Biological Sciences, Peptidoglycan binding, Biotechnology
Abstract

Background Endolysins of a number of bacteriophages, including coliphages T5, RB43, and RB49, target the peptidoglycans of the bacterial cell wall. The backbone of these bacterial peptidoglycans consist of alternating N-acetylglucosamine and N-acetylmuramic acid residues that is further “reinforced” by the peptide subunits. Because of the mesh-like structure and insolubility of peptidoglycans, the processes of the peptidoglycan binding and hydrolysis by enzymes cannot be studied by spectral methods. To overcome these issues we synthesized and analyzed here one of the simplest water soluble peptidoglycan mimetics. Methods A compound has been synthesized that mimics the peptidoglycan fragment of the bacterial cell wall, N-acetylglucosaminyl-β(1-4)-N-acetylmuramoyl-l-alanyl-γ-d-glutamyl-l-alanyl-d-alanine. NMR was used to study the degradation of this peptidoglycan mimetic by lytic l-alanoyl-d-glutamate peptidases of colibacteriophages T5, RB43, and RB49 (EndoT5, EndoRB43, and EndoRB49, respectively). Results The resulting glycopeptide mimetic was shown to interact with the studied enzymes. Its hydrolysis occurred through the bond between l-Ala and d-Glu. This artificial substrate mimetic was hydrolyzed by enzymes at different rates, which decreased outside the pH optimum. The EndoT5 demonstrated the lowest hydrolysis rate, whereas the EndoRB49-driven hydrolysis was the fastest one, and EndoRB43 displayed an intermediate potency. These observations are consistent with the hypothesis that EndoRB49 is characterized by the lowest selectivity, and hence the potentially broader spectrum of the peptidoglycan types subjected to hydrolysis, which was put forward in the previous study. We also show that to hydrolyze this glycopeptide mimetic, enzymes approach the glycopeptide near the methyl groups of all three alanines.

Published
2021

8. Comparative analysis of the active sites of orthologous endolysins of the Escherichia lytic bacteriophages T5, RB43, and RB49

Author

Svetlana I. Paskevich, Victor P. Kutyshenko, Dmitry A. Prokhorov, Galina V. Mikoulinskaia, Nikolai V. Molochkov, Vladimir N. Uversky, and Alexander Y. Yegorov

Subjects
inorganic chemicals, Circular dichroism, Stereochemistry, Proton Magnetic Resonance Spectroscopy, Lysin, 02 engineering and technology, Biochemistry, Active center, 03 medical and health sciences, Viral Proteins, Protein stability, Structural Biology, Escherichia, Catalytic Domain, Endopeptidases, Escherichia coli, Bacteriophages, Amino Acid Sequence, Molecular Biology, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, biology, Circular Dichroism, Active site, General Medicine, Hydrogen-Ion Concentration, 021001 nanoscience & nanotechnology, biology.organism_classification, Enzyme, Lytic cycle, chemistry, biology.protein, bacteria, Thermodynamics, 0210 nano-technology
Abstract

The methods of solution NMR, circular dichroism (CD), and differential scanning calorimetry (DSC) were used to study two zinc-containing L-alanyl-D-glutamate peptidases - endolysins of the pseudo T-even myoviruses RB43 and RB49 (EndoRB43 and EndoRB49, respectively), which are orthologous to the EndoT5, which is a zinc-containing L-alanyl-D-glutamate peptidase of the T5 siphovirus. The spatial conservation of the Zn2+-binding sites for the enzymes EndoT5, EndoRB43, and EndoRB49 was established, and the key role of Zn2+ ions in the stabilization of the spatial structures of these three peptidases was confirmed. We are showing here that the binding of the Zn2+ ion in the active center of EndoRB49 peptidase causes conformational rearrangements similar to those observed in the EndoT5 peptidase upon binding of Zn2+ and Ca2+ ions and lead to the formation of a catalytically active form of the enzyme. Therefore, the binding of the Zn2+ ion to the active site of EndoRB49 peptidase is a necessary and sufficient condition for functioning of this protein.

Published
2020

10. Structure and dynamics of the retro-form of the bacteriophage T5 endolysin

Author

Dmitry A. Prokhorov, Galina V. Mikoulinskaia, Victor P. Kutyshenko, Nikolai V. Molochkov, Sergei A. Taran, and Vladimir N. Uversky

Subjects
0301 basic medicine, 030103 biophysics, Protein Conformation, Phosphorylcholine, Detergents, Biophysics, Lysin, Polypeptide chain, Biochemistry, Micelle, Protein Structure, Secondary, Analytical Chemistry, Protein Aggregates, 03 medical and health sciences, Endopeptidases, Urea, Bacteriophages, Molecular Biology, Bacteriophage T5, Micelles, biology, Chemistry, Protein dynamics, Dynamics (mechanics), Nuclear magnetic resonance spectroscopy, biology.organism_classification, Crystallography, 030104 developmental biology, Protein folding, Peptides
Abstract

Using high-resolution NMR spectroscopy we conducted a comparative analysis of the structural and dynamic properties of the bacteriophage T5 endolysin (EndoT5) and its retro-form; i.e., a protein with the reversed direction of the polypeptide chain (R-EndoT5). We show that structurally, retro-form can be described as the molten globule-like polypeptide that is easily able to form large oligomers and aggregates. To avoid complications associated with this high aggregation propensity of the retro protein, we compared EndoT5 and R-EndoT5 in the presence of strong denaturants. This analysis revealed that these two proteins possess different internal dynamics in solutions containing 8M urea, with the retro-form being characterized by larger dimensions and slower internal dynamics. We also show that in the absence of denaturant, both forms of the bacteriophage T5 endolysin are able to interact with micelles formed by the zwitterionic detergent dodecylphosphocholine (DPC), and that the formation of the protein-micelle complexes leads to the significant structural rearrangement of polypeptide chain and to the formation of stable hydrophobic core in the R-Endo T5.

Published
2016
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11. Effect of C-terminal His-tag and purification routine on the activity and structure of the metalloenzyme, l-alanyl-d-glutamate peptidase of the bacteriophage T5

Author

Alexander Y. Yegorov, Galina V. Mikoulinskaia, Vladimir N. Uversky, Victor P. Kutyshenko, Sergei V. Chernyshov, and Dmitry A. Prokhorov

Subjects
Protein Folding, Cations, Divalent, Protein Conformation, Genetic Vectors, chemistry.chemical_element, Gene Expression, 02 engineering and technology, Zinc, Calcium, Biochemistry, Chromatography, Affinity, Catalysis, 03 medical and health sciences, chemistry.chemical_compound, Viral Proteins, Structural Biology, Nickel, Catalytic Domain, Protein purification, Endopeptidases, Enzyme Stability, Metalloproteins, Escherichia coli, Imidazole, Bacteriophages, Histidine, Cloning, Molecular, Molecular Biology, Bacteriophage T5, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, biology, Imidazoles, General Medicine, 021001 nanoscience & nanotechnology, biology.organism_classification, Recombinant Proteins, Folding (chemistry), Enzyme Activation, Enzyme, chemistry, 0210 nano-technology, Dialysis, Oligopeptides, Protein Binding
Abstract

In this work, we studied the effect of the C-terminally attached poly-histidine tag (His-tag), as well as the peculiarities of the protein purification procedure by the immobilized metal affinity chromatography (IMAC) on the activity and structure of the metalloenzyme, l-alanyl-d-glutamate peptidase of bacteriophage T5 (EndoT5), whose zinc binding site and catalytic aspartate are located near the C-terminus. By itself, His-tag did not have a significant effect on either activity or folding of the polypeptide chain, nor on the binding of zinc and calcium ions to the protein. However, the His-tagged EndoT5 samples had low shelf-life, with storage of these samples resulting in an increased propensity for protein self-association and decreased enzymatic activity of EndoT5. Furthermore, disastrous effects on the activity of the enzyme were exerted by the presence of imidazole and nickel ions accompanying metal chelate chromatography. The activity of the protein can be restored by thorough washing off of these low molecular impurities via the prolonged dialysis of the His-tagged EndoT5 samples at the specifically elaborated conditions.

Published
2018

12. High-resolution NMR structure of a Zn2+-containing form of the bacteriophage T5<scp>l</scp>-alanyl-<scp>d</scp>-glutamate peptidase

Author

Victor P. Kutyshenko, Nikolai V. Molochkov, Dmitry A. Prokhorov, Galina V. Mikoulinskaia, and Vladimir N. Uversky

Subjects
High resolution nmr, biology, Chemistry, General Chemical Engineering, General Chemistry, biology.organism_classification, Solution structure, D-Glutamate, Folding (chemistry), Crystallography, Hydrolase, Molecule, Amino acid residue, Bacteriophage T5
Abstract

This paper represents the spatial solution structure of the Zn2+-containing form of the bacteriophage T5 L-alanyl-D-glutamate peptidase (EndoT5-Zn2+). The core of this α + β protein is formed by three α-helices (residues 7–15, 20–30, and 87–104) and a β-sheet containing three β-strands (residues 35–39, 71–76, and 133–135). The protein has two short loops (residues 16–19 and 31–34), a medium-length loop (residues 77–86) containing a short β-hairpin (residues 77–82), and two long loops (residues 40–70 and 105–132). The long loops include a stable 310-helix (residues 66–68) and labile α-helices 46–53 and 113–117. Catalytic Zn2+-binding site is represented by three amino acid residues, His66, Asp73, and His133. The cation-binding His residues are located near the foundations of the long loops, whereas Asp73 is positioned in the middle of the core β-sheet. The catalytic center localization contributes to the stabilization of the entire molecule, with Zn2+-binding playing a key role in the folding of this protein.

Published
2015
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13. The study of the bacteriophage T5 deoxynucleoside monophosphate kinase active site by site-directed mutagenesis

Author

Yu. S. Skoblov, S. A. Taran, S. A. Feofanov, and Galina V. Mikoulinskaia

Subjects
chemistry.chemical_classification, biology, Arginine, Stereochemistry, Organic Chemistry, Mutagenesis, Active site, biology.organism_classification, Biochemistry, Amino acid, Enzyme, chemistry, biology.protein, Site-directed mutagenesis, Bacteriophage T5, Binding domain
Abstract

The amino acid residues essential for the enzymatic activity of bacteriophage T5 deoxyribonucleoside monophosphate kinase were determined using a computer model of the enzyme active site. By site-directed mutagenesis, cloning, and gene expression in E. coli, a series of proteins were obtained with single substitutions of the conserved active site amino acid residues—S13A, D16N, T17N, T17S, R130K, K131E, Q134A, G137A, T138A, W150F, W150A, D170N, R172I, and E176Q. After purification by ion exchange and affine chromatography electrophoretically homogeneous preparations were obtained. The study of the enzymatic activity with natural acceptors of the phosphoryl group (dAMP, dCMP, dGMP, and dTMP) demonstrated that the substitutions of charged amino acid residues of the NMP binding domain (R130, R172, D170, and E176) caused nearly complete loss of enzymatic properties. It was found that the presence of the OH-group at position 17 was also important for the catalytic activity. On the basis of the analysis of specific activity variations we assumed that arginine residues at positions 130 and 172 were involved in the binding to the donor γ-phosphoryl and acceptor α-phosphoryl groups, as well as the aspartic acid residue at position 16 of the ATP-binding site (P-loop), in the binding to some acceptors, first of all dTMP. Disproportional changes in enzymatic activities of partially active mutants, G137A, T138A, T17N, Q134A, S13A, and D16N, toward different substrates may indicate that different amino acid residues participate in the binding to various substrates.

Published
2013
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14. In vitro study of the antibacterial effect of the bacteriophage T5 thermostable endolysin on Escherichia coli cells

Author

Nikolai V. Molochkov, Andrei A. Zimin, Maria S. Shavrina, Galina V. Mikoulinskaia, Sergei V. Chernyshov, and Andrei V. Machulin

Subjects
0301 basic medicine, Lysis, 030106 microbiology, Lysin, Biology, Siphoviridae, medicine.disease_cause, Applied Microbiology and Biotechnology, Microbiology, 03 medical and health sciences, chemistry.chemical_compound, Viral Proteins, Endopeptidases, medicine, Escherichia coli, Bacteriophage T5, Polymyxin B, Protein Stability, Chlorhexidine, Drug Synergism, General Medicine, biology.organism_classification, Anti-Bacterial Agents, chemistry, Biochemistry, Gramicidin, Bacterial outer membrane, Bacteria, Biotechnology, medicine.drug
Abstract

Aims This study aimed to evaluate lysis of Escherichia coli stationary cell cultures induced by the combined action of bacteriophage T5 endolysin (l-alanyl-d-glutamate peptidase) and low doses of various cationic agents permeabilizing the outer membrane of Gram-negative bacteria (polymyxin B, gramicidin D, poly-l-lysine, chlorhexidine and miramistin). Methods and Results The enzyme activity was assayed with the turbidimetric method. Antimicrobial activity was assessed through the number of colony-forming units (CFUs); the results of calculation were represented as logarithmic units. The optical microscopy examination of bacterial cells was conducted in the phase-contrast mode. The use of bacteriophage T5 endolysin in combination with polymyxin B (0.4 μg ml-1) or chlorhexidine (0.5 μg ml-1) made it possible to reduce the number of colony-forming units by five orders of magnitude; and in combination with poly-l-lysine (80 μg ml-1) by four orders, as compared to control. The endolysin was found to be a thermostable protein: it retained ~65% of its initial activity after heating for 30-min at 90°C. Examining the curves of its thermal denaturation revealed the half-transition temperature to be 56.3 ± 1.0°C. Circular dichroism spectra showed that after recooling the protein restored up to 80% of its native structure. Conclusions A substantial synergistic effect of the bacteriophage T5 endolysin and membrane-permeabilizing compounds was demonstrated. Significance and Impact of the Study The study of thermal stability of the bacteriophage T5 endolysin and the quantitative assessment of its antimicrobial activity have been done for the first time. The approach examined lays foundations for designing a two-component preparation which would effectively lyse cells of Gram-negative pathogens from outside. This article is protected by copyright. All rights reserved.

Published
2016

15. Identification and characterization of the metal ion-dependent l-alanoyl-d-glutamate peptidase encoded by bacteriophage T5

Author

Galina V. Mikoulinskaia, S. A. Feofanov, Valentina Ya. Lysanskaya, O. A. Stepnaya, Irina V. Odinokova, and Andrei A. Zimin

Subjects
biology, Proteus vulgaris, Lysin, Pectobacterium carotovorum, Cell Biology, biology.organism_classification, medicine.disease_cause, Biochemistry, Microbiology, Lytic cycle, Hydrolase, medicine, Coliphage, Molecular Biology, Bacteriophage T5, Escherichia coli
Abstract

Although bacteriophage T5 is known to have lytic proteins for cell wall hydrolysis and phage progeny escape, their activities are still unknown. This is the first report on the cloning, expression and biochemical characterization of a bacteriophage T5 lytic hydrolase. The endolysin-encoding lys gene of virulent coliphage T5 was cloned in Escherichia coli cells, and an electrophoretically homogeneous product of this gene was obtained with a high yield (78% of total activity). The protein purified was shown to be an L-alanoyl-D-glutamate peptidase. The enzyme demonstrated maximal activity in diluted buffers (25-50 mM) at pH 8.5. The enzyme was strongly inhibited by EDTA and BAPTA, and fully reactivated by calcium/manganese chlorides. It was found that, along with E. coli peptidoglycan, peptidase of bacteriophage T5 can lyse peptidoglycans of other Gram-negative microorganisms (Pectobacterium carotovorum, Pseudomonas putida, Proteus vulgaris, and Proteus mirabilis). This endolysin is the first example of an L-alanoyl-D-glutamate peptidase in a virulent phage infecting Gram-negative bacteria. There are, however, a great many sequences in databases that are highly similar to that of bacteriophage T5 hydrolase, indicating a wide distribution of endolytic L-alanoyl-D-glutamate peptidases. The article discusses how an enzyme with such substrate specificity could be fixed in the process of evolution.

Published
2009
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16. Recent Origin of the Methacrylate Redox System in Geobacter sulfurreducens AM-1 through Horizontal Gene Transfer

Author

Galina V. Mikoulinskaia, Marina V. Zakharova, Alexander Galushko, O. V. Arkhipova, Fyodor A. Kondrashov, Margarita Meer, Vasilii K. Akimenko, and Томский государственный университет Институт биологии, экологии, почвоведения, сельского и лесного хозяйства (Биологический институт) Кафедра физиологии растений и биотехнологии

Subjects
метакрилатные системы, Evolució molecular, Gene Transfer, Horizontal, Operon, Sequence analysis, Science, Molecular Sequence Data, Genome, гены, Bacterial Proteins, Molecular evolution, Gene Order, Geobacter sulfurreducens, Gene, Phylogeny, Genetics, Multidisciplinary, biology, food and beverages, biology.organism_classification, горизонтальный перенос генов, Biological Evolution, Horizontal gene transfer, Medicine, Methacrylates, Geobacter, Oxidation-Reduction, Genome, Bacterial, Research Article
Abstract

The origin and evolution of novel biochemical functions remains one of the key questions in molecular evolution. We study recently emerged methacrylate reductase function that is thought to have emerged in the last century and reported in Geobacter sulfurreducens strain AM-1. We report the sequence and study the evolution of the operon coding for the flavin-containing methacrylate reductase (Mrd) and tetraheme cytochrome с (Mcc) in the genome of G. sulfurreducens AM-1. Different types of signal peptides in functionally interlinked proteins Mrd and Mcc suggest a possible complex mechanism of biogenesis for chromoproteids of the methacrylate redox system. The homologs of the Mrd and Mcc sequence found in δ-Proteobacteria and Deferribacteres are also organized into an operon and their phylogenetic distribution suggested that these two genes tend to be horizontally transferred together. Specifically, the mrd and mcc genes from G. sulfurreducens AM-1 are not monophyletic with any of the homologs found in other Geobacter genomes. The acquisition of methacrylate reductase function by G. sulfurreducens AM-1 appears linked to a horizontal gene transfer event. However, the new function of the products of mrd and mcc may have evolved either prior or subsequent to their acquisition by G. sulfurreducens AM-1. The work has been supported by a grant of the HHMI International Early Career Scientist Program (55007424), the Spanish Ministry of Economy and Competitiveness (EUI-EURYIP-2011-4320) as part of the EMBO YIP program, two grants from the Spanish Ministry of Economy and Competitiveness, "Centro de Excelencia Severo Ochoa 2013–2017 (Sev-2012-0208)" and (BFU2012-31329), the European Union and the European Research Council under grant agreement/n335980_EinME.

Published
2015

17. Identification, cloning, and expression of bacteriophage T5 dnk gene encoding a broad specificity deoxyribonucleoside monophosphate kinase (EC 2.7.4.13)

Author

Andrei A. Zimin, S. A. Feofanov, A. I. Miroshnikov, and Galina V. Mikoulinskaia

Subjects
Models, Molecular, Molecular Sequence Data, Gene Expression, Genome, Viral, medicine.disease_cause, law.invention, chemistry.chemical_compound, law, medicine, Amino Acid Sequence, Cloning, Molecular, Bacteriophage T5, Gene, Escherichia coli, Peptide sequence, Phosphotransferases (Phosphate Group Acceptor), Base Sequence, biology, Nucleic acid sequence, Sequence Analysis, DNA, Physical Chromosome Mapping, biology.organism_classification, Molecular biology, Recombinant Proteins, Protein Structure, Tertiary, Deoxyribonucleoside, Biochemistry, chemistry, GenBank, Recombinant DNA, T-Phages, Plasmids, Biotechnology
Abstract

The nucleotide sequence corresponding to 13–19.5% of the bacteriophage T5 genome in early region C was determined (GenBank AY140897 ). One of the five major single-stranded interruptions (nicks) of bacteriophage T5 DNA was identified at 18.5%. The sequenced region was annotated and the putative functions of some open reading frames were proposed by comparison with databases. The dnk gene, encoding a deoxyribonucleoside monophosphate kinase, was identified using a previously defined N-terminal amino acid sequence. The gene was cloned and expressed in Escherichia coli , the enzyme was purified to homogeneity with high yield using two alternative methods, and the recombinant deoxyribonucleoside monophosphate kinase was found to have the same activity and specificity as the native enzyme.

Published
2004
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18. A new broad specificity deoxyribonucleoside monophosphate kinase encoded by gene 52 of phage ϕC31

Author

Andrei A. Zimin, A. I. Miroshnikov, S. A. Feofanov, and Galina V. Mikoulinskaia

Subjects
Kinase, Biophysics, Bacillus Phages, General Chemistry, General Medicine, MAP3K7, Biochemistry, MAP3K8, Streptomyces, Substrate Specificity, Deoxyribonucleoside, Phosphotransferases (Alcohol Group Acceptor), chemistry.chemical_compound, Bacteriolysis, chemistry, Escherichia coli, Cloning, Molecular, Gene
Published
2007
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20. Contributors

Author

Catherine Anne Abbott, Carmela R. Abraham, Hideki Adachi, Osao Adachi, Zach Adam, Michael W.W. Adams, Michael J. Adang, Ibrahim M. Adham, Patrizia Aducci, David A. Agard, Alexey A. Agranovsky, Tetsuya Akamatsu, Yoshinori Akiyama, Reidar Albrechtsen, Alí Alejo, Sean M. Amberg, Alexander Y. Amerik, Piti Amparyup, Felipe Andrade, Germán Andrés, Daniel M. Andrews, Robert K. Andrews, Toni M. Antalis, Colin S. Anthony, Naoya Aoki, Suneel S. Apte, Kazunari Arima, Gérard Arlaud, Raghuvir Krishnaswamy Arni, Pascal Arnoux, Nathan N. Aronson, Michel Arthur, Yasuhisa Asano, Paolo Ascenzi, Marina T. Assakura, David S. Auld, Veridiana de Melo Rodrigues Ávila, Francesc X. Avilés, William M. Awad, Anand K. Bachhawat, Shan Bai, Teaster T. Baird, S. Paul Bajaj, Susan C. Baker, Agnieszka Banbula, Alan J. Barrett, Jemima Barrowman, John D. Bartlett, Jörg W. Bartsch, Nikola Baschuk, Isolda P. Baskova, Jyotsna Batra, Karl Bauer, Ulrich Baumann, Wolfgang Baumeister, Cédric Bauvois, Alex Bayés, Anne Beauvais, Christoph Becker-Pauly, Tadhg P. Begley, Miklós Békés, Robert Belas, Daniah Beleford, Teruhiko Beppu, Ernst M. Bergmann, Bruno A. Bernard, Dominique Bernard, Michael C. Berndt, Giovanna Berruti, Colin Berry, Greg P. Bertenshaw, Christian Betzel, Chetana Bhaskarla, Manoj Bhosale, Gabriele Bierbaum, B. Bjarnason Jón, Michael Blaber, Michael J. Blackman, Alexander Blinkovsky, Jef D. Boeke, Matthew Bogyo, Stefan Bohn, Guy Boileau, Mike Boland, Tové C. Bolken, Judith S. Bond, Jan Bondeson, Javier Bordallo, Claudia Borelli, Tiago O. Botelho, Richard R. Bott, David G. Bourne, Niels Bovenschen, Ralph A. Bradshaw, Klaus Breddam, Keith Brew, Paul J. Brindley, Diane L. Brinkman, Collette Britton, Jeff R. Broadbent, Anne Broadhurst, Dieter Brómme, Murray Broom, Jeremy S. Brown, Mark A. Brown, Iris Bruchhaus, Barbara A. Burleigh, Kristin E. Burns, James F. Burrows, Michael J. Butler, David J. Buttle, Chelsea M. Byrd, Tony Byun, Sandrine Cadel, Conor R. Caffrey, Santiago Cal, Javier Caldentey, Thomas Candela, Clemente Capasso, Daniel R. Capriogilio, Vincenzo Carginale, Adriana Karaoglanovic Carmona, Vern B. Carruthers, Francis J. Castellino, Joseph J. Catanese, Bruce Caterson, George H. Caughey, Naimh X. Cawley, Tim E. Cawston, Juan José Cazzulo, Jijie Chai, Karl X. Chai, Olga Meiri Chaim, L.S. Chang, Julie Chao, Marie-Pierre Chapot-Chartier, Jean-Louis Charli, Paulette Charlier, Karen J. Chave, Jian-Min Chen, Jinq-May Chen, Li-Mei Chen, Ya-Wen Chen, Yu-Yen Chen, Bernard Chevrier, Jean-François Chich, Jeremy Chien, Suneeta Chimalapati, Ki Joon Cho, Kwan Yong Choi, Woei-Jer Chuang, Chin Ha Chung, Ivy Yeuk Wah Chung, Christine Clamagirand, Ian M. Clark, Adrian K. Clarke, Nicola E. Clarke, Steven Gerard Clarke, Philippe Clauziat, Judith A. Clements, Catherine Coffinier, Paul Cohen, Alain Colige, Anne Collignon, Sean D. Colloms, Andreas Conzelmann, Graham H. Coombs, Jakki C. Cooney, Jonathan B. Cooper, Max D. Cooper, Nikki A. Copeland, Graeme S. Cottrell, Joseph T. Coyle, Charles S. Craik, John W.M. Creemers, Daniela Cretu, Jenifer Croce, Keith J. Cross, Rosario Cueva, Sheng Cui, Luis Cunha, Simon Cutting, Christophe d’Enfert, Hugues D’Orchymont, Björn Dahlbäck, Shujia Dai, Ross E. Dalbey, John P. Dalton, Pam M. Dando, R.M. Daniel, Sergei M. Danilov, Donna E. Davies, Heloisa S. De Araujo, Teresa De los Santos, Viviana de Luca, Ingrid De Meester, Ana Karina de Oliveira, Eduardo Brandt de Oliveira, Pedro Lagerblad De Oliveira, Sarah de Vos, Jeroen Declercq, Wim Declercq, Ala-Eddine Deghmane, Niek Dekker, Sonia Del Prete, Marina Del Rosal, Bernard Delmas, Robert DeLotto, Ilya V. Demidyuk, Mark R. Denison, Jan M. Deussing, Lakshmi A. Devi, Eleftherios P. Diamandis, Isabel Diaz, Araceli Díaz-Perales, Bauke W. Dijkstra, Yan Ding, Jack E. Dixon, Johannes Dodt, Terje Dokland, Iztok Dolenc, Ningzheng Dong, Tran Cat Dong, Ying Dong, Mitesh Dongre, Mark Donovan, Timothy M. Dore, Loretta Dorstyn, Hong Dou, Zhicheng Dou, Annette M. Dougall, Marcin Drag, Edward G. Dudley, Ben M. Dunn, Bruno Dupuy, Maria Conceicāo Duque-Magalhāes, M. Asunción Durá, Yves Eeckhout, Vincent Eijsink, Arthur Z. Eisen, Azza Eissa, Sandra Eklund, Ziad M. Eletr, Vincent Ellis, Wolfgang Engel, Ervin G. Erdös, Teresa Escalante, David A. Estell, Michael Etscheid, Herbert J. Evans, Roger D. Everett, Alex C. Faesen, Falk Fahrenholz, Miriam Fanjul-Fernández, Christopher J. Farady, Georges Feller, Hong Feng, Kurt M. Fenster, Claude Férec, Silvia Ferrari, Barbara Fingleton, Jed F. Fisher, Paula M. Fives-Taylor, Loren G. Fong, F. Forneris, Brian M. Forster, Friedrich Forster, Simon J. Foster, Thierry Foulon, Stephen I. Foundling, Jay William Fox, Bruno Franzetti, Alejandra P. Frasch, Hudson H. Freeze, Jean-Marie Frère, Teryl K. Frey, Beate Fricke, Lloyd D. Fricker, Rafael Fridman, Christopher J. Froelich, Camilla Fröhlich, Hsueh-Liang Fu, Cynthia N. Fuhrmann, Satoshi Fujimura, Hiroshi Fujiwara, Jun Fukushima, Keiichi Fukuyama, Robert S. Fuller, Martin Fusek, Christine Gaboriaud, Christian Gache, Oleksandr Gakh, Peter Gal, Junjun Gao, Adolfo García-Sastre, Donald L. Gardiner, John A. Gatehouse, G.M. Gaucher, Francis Gauthier, Jean-Marie Ghuysen, Wade Gibson, Jennifer Gillies, Elzbieta Glaser, Fabian Glaser, Michael H. Glickman, Peter Goettig, Colette Goffin, Eiichi Gohda, Alfred L. Goldberg, Daniel E. Goldberg, Gregory I. Goldberg, Nathan E. Goldfarb, F. Xavier Gomis-Rüth, B. Gopal, Alexander E. Gorbalenya, Stuart G. Gordon, Mark D. Gorrell, Friedrich Götz, Theodoros Goulas, Cécile Gouzy-Darmon, K. Govind, Lászlo Gráf, Robert R. Granados, Melissa Ann Gräwert, Douglas A. Gray, Thomas P. Graycar, Jonathan A. Green, Luiza Helena Gremski, Michael Groll, Tania Yu Gromova, P. Gros, Marvin J. Grubman, Amy M. Grunden, Ágústa Gudmundsdóttir, Micheline Guinand, Djamel Gully, Alla Gustchina, José María Gutiérrez, Byung Hak Ha, Jesper Z. Haeggström, James H. Hageman, Johanna Haiko, Stephan Hailfinger, Hans Michael Haitchi, Ji Seon Han, Chantal Hanquez, Minoru Harada, Ikuko Hara-Nishimura, Marianne Harboe, Torleif Härd, David A. Harris, Ulrich Hassiepen, Shoji Hata, Akira Hattori, Rong-Qiao He, Albert J.R. Heck, Dirk F. Hendricks, Bernhard Henrich, Patrick Henriet, Andrés Hernández-Arana, Irma Herrera-Camacho, Gerhard Heussipp, Toshihiko Hibino, P.M. Hicks, Bradley I. Hillman, B. Yukihiro Hiraoka, Jun Hiratake, Yohei Hizukuri, Heng-Chien Ho, Ngo Thi Hoa, Mark Hochstrasser, Kathryn M. Hodge, Theo Hofmann, Thomas Hohn, John R. Hoidal, Joachim-Volker Höltje, Koichi J. Homma, John F. Honek, Vivian Y.H. Hook, John D. Hooper, Nigel M. Hooper, Kazuo Hosoi, Christopher J. Howe, Dennis E. Hruby, James J.-D. Hseih, Chun-Chieh Hsu, Tony T. Huang, Tur-Fu Huang, Yoann Huet, Clare Hughes, Jean-Emmanuel Hugonnet, Adrienne L. Huston, Oumaïma Ibrahim-Granet, Eiji Ichishima, Yukio Ikehara, Tadashi Inagami, Jessica Ingram, R.E. Isaac, Grazia Isaya, Clara E. Isaza, Shin-ichi Ishii, Amandine Isnard, Kiyoshi Ito, Koreaki Ito, Yoshifumi Itoh, Xavier Iturrioz, Sadaaki Iwanaga, Ralph W. Jack, Mel C. Jackson, Michael N.G. James, Jiří Janata, Claire Janoir, Hanna Janska, Ken F. Jarrell, Mariusz Jaskolski, Sheila S. Jaswal, Ying Y. Jean, Dieter E. Jenne, Young Joo Jeon, Ping Jiang, John E. Johnson, Michael D. Johnson, James A. Johnston, Amanda Jones, Elizabeth W. Jones, Carine Joudiou, Luiz Juliano, Hea-Jin Jung, Ray Jupp, Todd F. Kagawa, Hubert Kalbacher, Yayoi Kamata, Shuichi Kaminogawa, Yoshiyuki Kamio, Makoto Kaneda, Sung Gyun Kang, Sung Hwan Kang, Mary Kania, Tomasz Kantyka, Nobuyuki Kanzawa, Abdulkarim Y. Karim, Takafumi Kasumi, Hiroaki Kataoka, Hardeep Kaur, Shun-Ichiro Kawabata, Mari Kawaguchi, John Kay, Murat Kaynar, Kenneth C. Keiler, R.M. Kelly, Nathaniel T. Kenton, Michael A. Kerr, Kristof Kersse, Jukka Kervinen, Benedikt M. Kessler, Efrat Kessler, Timo K. Khoronen, Simon Kidd, Marjolein Kikkert, Mogens Kilian, Do-Hyung Kim, Doyoun Kim, Eunice EunKyeong Kim, In Seop Kim, Jung-Gun Kim, Kyeong Kyu Kim, Kyung Hyun Kim, Matthew S. Kimber, Yukio Kimura, Heidrun Kirschke, Yoshiaki Kiso, Colin Kleanthous, Jürgen R. Klein, Michael Klemba, Beata Kmiec, Hideyuki Kobayashi, Hiroyuki Kodama, Gerald Koelsch, Jan Kok, P.E. Kolattukody, Fabrice A. Kolb, Harald Kolmar, Yumiko Komori, Jan Konvalinka, Brice Korkmaz, Sergey V. Kostrov, Hans-Georg Kräusslich, Gabi Krczal, Lawrence F. Kress, Magnüs Már Kristjánsson, Tomáš Kučera, Sayali S. Kukday, Hidehiko Kumagai, Sharad Kumar, Malika Kumarasiri, Takashi Kumazaki, Beate M. Kümmerer, Kouji Kuno, Markku Kurkinen, Eva Kutejová, Marie Kveiborg, Agnieszka Kwarciak, Liisa Laakkonen, Nikolaos E. Labrou, Gavin D. Laing, Gayle Lamppa, Thomas Langer, Richard A. Laursen, Richard A. Lawrenson, Matthew D. Layne, Bernard F. Le Bonniec, María C. Leal, Ronald M. Lechan, David H. Lee, Irene Lee, Jae Lee, Kye Joon Lee, Soohee Lee, Xiaobo Lei, Jonathan Leis, Ellen K. LeMosy, Thierry Lepage, Stephen H. Leppla, Adam Lesner, Ivan A.D. Lessard, Guy Lhomond, Huilin Li, Shu-Ming Li, Weiguo Li, Ta-Hsiu Liao, Robert C. Liddington, Toby Lieber, H.R. Lijnen, Christopher D. Lima, Chen-Yong Lin, Gang Lin, Ming T. Lin, Xinli Lin, Yee-Shin Lin, L.L. Lindsay, William N. Lipscomb, John W. Little, Ching-Chuan Liu, Chuan-ju Liu, Mark O. Lively, Nurit Livnat-Levanon, Per O. Ljungdahl, Catherine Llorens-Cortes, Peter Lobel, Y. Peng Loh, Jouko Lohi, G.P. Lomonossoff, Yvan Looze, Carlos López-Otin, Landys Lopez-Quezada, Alex Loukas, Long-Sheng Lu, Áke Lundwall, Liu-Ying Luo, Andrei Lupas, Dawn S. Luthe, Nicholas J. Lynch, Peter J. Lyons, Vivian L. MacKay, Jesica M. Levingston Macleod, Viktor Magdolen, Jean-Luc Mainardi, Kauko K. Mäkinen, Jeremy P. Mallari, Surya P. Manandhar, Fajga R. Mandelbaum, Anne M. Manicone, Johanna Mansfeld, Joseph Marcotrigiano, Michael Mares, Gemma Marfany, Francis S. Markland, Judith Marokházi, Hélène Marquis, Robert A. Marr, Enzo Martegani, Erik W. Martin, Manuel Martinez, L. Miguel Martins, Masato Maruyama, Masugi Maruyama, Sususmu Maruyama, Takeharu Masaki, Ramin Massoumi, Rency T. Mathew, Lynn M. Matrisian, Yoshihiro Matsuda, Osamu Matsushita, Marco Matuschek, Anna Matušková, Krisztina Matúz, Cornelia Mauch, Michael R. Maurizi, Lorenz M. Mayr, Dewey G. McCafferty, J. Ken McDonald, James H. McKerrow, David McMillan, Robert P. Mecham, Darshini P. Mehta, Chris Meisinger, Alan Mellors, Roger G. Melton, Jeffrey A. Melvin, Robert Ménard, Luis Menéndez-Arias, Milene C. Menezes, Andrew Mesecar, Stéphane Mesnage, Diane H. Meyer, Gregor Meyers, Susan Michaelis, Karolina Michalska, Wojciech P. Mielicki, Igor Mierau, Galina V. Mikoulinskaia, Charles G. Miller, Lydia K. Miller, John Mills, Kenneth V. Mills, Jinrong Min, Michel-Yves Mistou, Yoshio Misumi, Shin-ichi Miyoshi, Shigehiko Mizutani, Shahriar Mobashery, Satsuki Mochizuki, William L. Mock, Frank Möhrlen, Nathalie Moiré, Paul E. Monahan, Angela Moncada-Pazos, Véronique Monnet, Michel Monod, Cesare Montecucco, Laura Morelli, Sumiko Mori, Takashi Morita, James H. Morrissey, Richard J. Morse, John S. Mort, Uffe H. Mortensen, Rory E. Morty, Joel Moss, Hidemasa Motoshima, Jeremy C. Mottram, Ana M. Moura-da-Silva, Mary Beth Mudgett, Egbert Mundt, Kazuo Murakami, Mario Tyago Murakami, Kimiko MurakamiMurofoshi, Sawao Murao, Gillian Murphy, M.R.N. Murthy, Tatsushi Muta, Elmarie Myburgh, Nino Mzhavia, A.H.M. Nurun Nabi, Hideaki Nagase, Michael W. Nagle, Dorit K. Nägler, Rajesh R. Naik, Divya B. Nair, Toshiki Nakai, Yoshitaka Nakajima, Yukio Nakamura, Hitoshi Nakatogawa, Toru Nakayama, Natalia N. Nalivaeva, Dipankar Nandi, Maria Clara Leal Nascimento-Silva, Kim Nasmyth, Carl F. Nathan, Fernando Navarro-García, Dayane Lorena Naves, Danny D. Nedialkova, Keir C. Neuman, Jeffrey-Tri Nguyen, Ky-Anh Nguyen, Gabriela T. Niemirowicz, Toshiaki Nikai, Eiichiro Nishi, Wataru Nishii, Makoto Nishiyama, Yasuhiro Nishiyama, Masatoshi Noda, Seiji Nomura, Shigemi Norioka, Desire M.M. Nsangou, Amornrat O’Brien, Michael B. O’Connor, Kohei Oda, Irina V. Odinokova, Joyce Oetjen, Teru Ogura, Dennis E Ohman, Yoshinori Ohsumi, Mukti Ojha, Akinobu Okabe, Yasunori Okada, Keinosuke Okamoto, Kenji Okuda, Nobuaki Okumura, Takashi Okuno, Kjeld Oleson, Priscila Oliveira de Giuseppe, Martin Olivier, Yasuko Ono, Stephen Oroszlan, Nobuyuki Ota, Michael Ovadia, Jiyang O-Wang, Claus Oxvig, Jeremy C.L. Packer, Sergio Padilla-López, Mark Paetzel, Michael J. Page, Andrea Page-McCaw, Mark J.I. Paine, Byoung Chul Park, Eunyong Park, John E. Park, Pyong Woo Park, Sung Goo Park, Kirk L. Parkin, William C Parks, Thaysa Paschoalin, Annalisa Pastore, Alexander Nikolich Patananan, Sudhir Paul, Henry L. Paulson, Ulrich von Pawel-Rammingen, David A. Pearce, Mark S. Pearson, Duanqing Pei, Gunnar Pejler, Alan D. Pemberton, Jianhao Peng, Julien Pernier, Jan-Michael Peters, Thorsten Pfirrmann, Viet-Laï Pham, Iva Pichová, Darren Pickering, Christophe Piesse, David Pignol, Robert N. Pike, Lothaire Pinck, Hubert Pirkle, Henry C. Pitot, Andrew G. Plaut, Hidde Ploegh, László Polgár, Corrine Porter, Rolf Postina, Jan Potempa, Knud Poulsen, Scott D. Power, Rex. F. Pratt, Gerd Prehna, Gilles Prévost, Alexey V. Pshezhetsky, Mohammad A. Qasim, Feng Qian, Jiazhou Qiu, Víctor Quesada, Evette S. Radisky, Stephen D. Rader, Kavita Raman, Andrew J. Ramsay, Derrick E. Rancourt, Najju Ranjit, Narayanam V. Rao, Kiira Ratia, Neil D. Rawlings, Robert B. Rawson, Vijay Reddy, Colvin M. Redman, Maria Elena Regonesi, Andreas S. Reichert, Antonia P. Reichl, Han Remaut, S. James Remington, Martin Renatus, David Reverter, Eric C. Reynolds, Mohamed Rholam, Charles M. Rice, Todd W. Ridky, Howard Riezman, D.C. Rijken, Marie-Christine Rio, Alison Ritchie, Janine Robert-Baudouy, Mark W. Robinson, Michael Robinson, Adela Rodriguez-Romero, Renata Santos Rodriques, John C. Rogers, Camilo Rojas, Floyd E. Romesberg, David J. Roper, Nora Rosas-Murrieta, A.M. Rose, Philip J. Rosenthal, J. Rosing, Ornella Rossetto, Véronique Rossi, Richard A. Roth, Hanspeter Rottensteiner, Andrew D. Rowan, Mikhail Rozanov, Alexandra Rucavado, Andrea Ruecker, Françoise Rul, Till Rümenapf, Ilaria Russo, Martin D. Ryan, Elena Sacco, J. Evan Sadler, W. Saenger, Hans-Georg Sahl, Mohammed Sajid, Masayoshi Sakaguchi, Fumio Sakiyama, Maria L. Salas, Maria Cristina O. Salgado, Guy S. Salvesen, Edith Sánchez, Eladio F. Sanchez, Qing-Xiang Amy Sang, Krishnan Sankaran, Susanta K. Sarkar, Michael P. Sarras, Yoshikiyo Sasagawa, Araki Satohiko, Eric Sauvage, Loredana Saveanu, H.S. Savithri, Hitoshi Sawada, R. Gary Sawers, Isobel A. Scarisbrick, Andreas Schaller, Justin M. Scheer, Friedrich Scheiflinger, Cordelia Schiene-Fischer, Uwe Schlomann, Manfred Schlösser, Alvin H. Schmaier, Walter K. Schmidt, Anette Schneemann, Rick G. Schnellmann, Henning Scholze, Lutz Schomburg, Wilhelm J. Schwaeble, Christopher J. Scott, Rosaria Scudiero, Atsuko Sehara-Fujisawa, Nabil G. Seidah, Motoharu Seiki, Junichi Sekiguchi, Andrea Senff-Ribeiro, Ihn Sik Seong, Mihaela Serpe, Solange M.T. Serrano, Peter Setlow, Tina Shahian, M. Shanks, Feng Shao, Steven D. Shapiro, Navneet Sharma, Lindsey N. Shaw, Aimee Shen, Lei Shen, Roger F. Sherwood, Yun-Bo Shi, Hitoshi Shimoi, Yoichiro Shimura, A.D. Shirras, Viji Shridhar, Jinal K. Shukla, Ene Siigur, Jüri Siigur, Natalie C. Silmon de Monerri, Robert B. Sim, James P. Simmer, William H. Simmons, Jaspreet Singh, Alison Singleton, Tatiana D. Sirakova, Titia K. Sixma, Tim Skern, Randal A. Skidgel, Jeffrey Slack, David E. Sleat, Barbara S. Slusher, Janet L. Smith, Matthew A. Smith, Mark J. Smyth, Erik J. Snijder, Solmaz Sobhanifar, Kenneth Söderhaäll, Istvan Sohar, Peter Sonderegger, Marcos Henrique Ferreira Sorgine, Hiroyuki Sorimachi, Karen E. Soukhodolets, Tatiana de Arruda Campos Brasil de Souza, Tamás Sperka, Shiranee Sriskandan, Joseph W. St. Geme, Raymond J. St. Leger, Peter Staib, James L. Steele, Bjarki Stefansson, Christian Steinkühler, Leisa M. Stenberg, Johan Stenflo, Henning R. Stennicke, Valentin M. Stepanov, Olga A. Stepnaya, Frank Steven, Richard L. Stevens, Kenneth J. Stevenson, Mathieu St-Louis, Christopher C. Stobart, Walter Stöcker, Andrew C. Storer, Norbert Sträter, Ellen G. Strauss, James H. Strauss, Kvido Stříšovský, Natalie C.J. Strynadka, Edward D. Sturrock, Dan Su, Xiao-Dong Su, Paz Suárez-Rendueles, Traian Sulea, Venkatesh Sundararajan, Ryoji Suno, Carolyn K. Suzuki, Fumiaki Suzuki, Hideyuki Suzuki, Nobuhiro Suzuki, Stephen Swenson, Rose L. Szabady, Pal Bela Szecsi, Lászlo Szilágyi, Muhamed-Kheir Taha, Eizo Takahashi, Kenji Takahashi, Toshiro Takai, Atsushi Takeda, Soichi Takeda, Jeremy J.R.H. Tame, Tomohiro Tamura, Fulong Tan, Keiji Tanaka, Carmen Tanase, Jordan Tang, Martha M. Tanizaki, Egbert Tannich, Guido Tans, Anthony L. Tarentino, Anchalee Tassanakajon, Hiroki Tatsumi, Norbert Tautz, Erin Bassford Taylor, Pedro Filipe Teixeira, Bhanu Prakash V.L. Telugu, Markus F. Templin, Shigeyuki Terada, Uchikoba Tetsuya, C. Thacker, Maulik Thaker, Heinz-Jürgen Thiel, Nicole Thielens, Gonzales Thierry, Karine Thivierge, Mark D. Thomas, Margot Thome, Mary K. Thorsness, Peter E. Thorsness, Natalie J. Tigue, Sokol V. Todi, Birgitta Tomkinson, Fiorella Tonello, Liang Tong, H.S. Toogood, Paolo Tortora, József Tözsèr, Luiz Rodolpho Travassos, James Travis, Dilza Trevisan-Silva, Francesca Trinchella, Neil N. Trivedi, Carol M. Troy, Harald Tschesche, Yu-Lun Tseng, Masafumi Tsujimoto, Anthony T. Tu, Kathleen E. Tumelty, Boris Turk, Dusan Turk, Vito Turk, Anthony J. Turner, Tetsuya Uchikoba, Takayuki Ueno, Alejandro P. Ugalde, Veli-Jukka Uitto, Sinisa Urban, Olivier Valdenaire, Adrian Valli, Jozef Van Beeumen, Bertus Van den Burg, Renier A.L. Van der Hoorn, Jan Maarten van Dijl, Peter Van Endert, Bram J. Van Raam, Harold E. Van Wart, Tom Vanden Berghe, Peter Vandenabeele, Margo Vanoni, Silvio Sanches Veiga, William H. Velander, Gloria Velasco, Josep Vendrell, I. István Venekei, Vaclav Vetvicka, F.-Nora Vögtle, Waldemar Vollmer, Kei Wada, Fred W. Wagner, Sun Nyunt Wai, Timothy Wai, Shane Wainwright, Kenneth W. Walker, Stephen J. Walker, Jean Wallach, Linda L. Walling, Peter N. Walsh, Hai-Yan Wang, Hengbin Wang, Jianwei Wang, Peng Wang, Ping Wang, Michael Wassenegger, Kunihiko Watanabe, Helen Webb, Joseph M. Weber, Niklas Weber, Daniel R. Webster, Shuo Wei, Rodney A. Welch, James A. Wells, Herbert Wenzel, Ingrid E. Wertz, Ulla W. Wewer, Alison R. Whyteside, Sherwin Wilk, Jean-Marc Wilkin, Claudia Wilmes, Jakob R. Winther, David S. Wishart, Alexander Wlodawer, J. Fred Woessner, Michael S. Wolfe, Wilson Wong, Roger Woodgate, Gerry Wright, Jiunn-Jong Wu, Qingyu Wu, Magdalena Wysocka, Chao Xu, Zhenghong Xu, Kinnosuke Yahori, Shoji Yamada, Nozomi Yamaguchi, Shinji Yamaguchi, Yoshio Yamakawa, Hiroki Yamamoto, Ikao Yana, Maozhou Yang, Na Yang, Chenjuan Yao, Tingting Yao, Noriko Yasuda, Toshimasa Yasuhara, Shigeki Yasumasu, Edward T.H. Yeh, Irene Yiallouros, Jiang Yin, Hiroo Yonezawa, Soon Ji Yoo, Tadashi Yoshimoto, Michael W. Young, Stephen G. Young, Nousheen Zaidi, Ludmila L. Zavalova, Peter Zavodszky, Aidong Zhang, Xianming Zhang, Yi-Zheng Zhang, Dominick Zheng, Guangming Zhong, Rong Zhong, Yuan Zhou, Zhaohui Sunny Zhou, Michael Zick, Paola Zigrino, and Andrei A. Zimin

Published
2013
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21. [Substrate specificity of T5 bacteriophage deoxyribonucleoside monophosphate kinase and its application for the synthesis of [alpha-(32)P]d/rNTP]

Author

Yu. S. Skoblov, S. A. Feofanov, Galina V. Mikoulinskaia, A. Yu. Skoblov, S. A. Taran, and A. I. Miroshnikov

Subjects
inorganic chemicals, macromolecular substances, Siphoviridae, environment and public health, Biochemistry, Substrate Specificity, Bacteriophage, chemistry.chemical_compound, Viral Proteins, RNTP, Bioorganic chemistry, Bacteriophage T5, chemistry.chemical_classification, Phosphotransferases (Phosphate Group Acceptor), biology, Kinase, Nucleotides, Organic Chemistry, Phosphorus Isotopes, biology.organism_classification, Deoxyribonucleoside, enzymes and coenzymes (carbohydrates), Enzyme, chemistry, Isotope Labeling, bacteria, Phosphorylation
Abstract

Bacteriophage T5 deoxynucleoside monophosphate kinase (dNMP kinase, EC 2.7.4.13) is shown to catalyze the phosphorylation of both d2CMP and ribonucleotides AMP, GMP, and CMP, but does not phosphorylate UMP. For natural acceptors of the phosphoryl group, k m and k cat were found. The applicability of T5 dNMP kinase as a universal enzyme capable of the phosphorylation of labelled r/dNMP was shown for the synthesis of [α-32P]rNTP and [α-32P]dNTP.

Published
2010

22. Purification and characterization of the deoxynucleoside monophosphate kinase of bacteriophage T5

Author

Sergei I Gubanov, S. A. Feofanov, Andrei A. Zimin, A. I. Miroshnikov, Igor V Kolesnikov, and Galina V. Mikoulinskaia

Subjects
Time Factors, Deoxyribonucleotides, Molecular Sequence Data, Biology, medicine.disease_cause, medicine, Escherichia coli, Bacteriophages, Amino Acid Sequence, Peptide sequence, Polyacrylamide gel electrophoresis, Bacteriophage T5, Anion Exchange Resins, chemistry.chemical_classification, Phosphotransferases (Phosphate Group Acceptor), Molecular mass, Hydrogen-Ion Concentration, biology.organism_classification, Chromatography, Ion Exchange, Molecular biology, Amino acid, Protein Structure, Tertiary, Resins, Synthetic, Enzyme, Isoelectric point, chemistry, Biochemistry, Models, Chemical, Ammonium Sulfate, Electrophoresis, Polyacrylamide Gel, Ultracentrifugation, Software, Biotechnology
Abstract

Deoxynucleoside monophosphate kinase (dNMP kinase) of bacteriophage T5 (EC 2.7.4.13) was purified to apparent homogeneity from phage-infected Escherichia coli cells. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gel showed that the enzyme has a molecular mass of about 29 kDa. The molecular mass of dNMP kinase estimated by analytical equilibrium ultracentrifugation turned out to be 29.14 +/- 3.03 kDa. These data suggest that the enzyme exists in solution as a monomer. The isoelectric point of dNMP kinase was found to be 4.2. The N-terminal amino acid sequence, comprising 21 amino acids, was determined to be VLVGLHGEAGSGKDGVAKLII. A comparison of this amino acid sequence and those of known enzymes with a similar function suggests the presence of a nucleotide-binding site in the sequenced region.

Published
2003

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