10 results on '"Raulinaitis V"'
Search Results
2. Identification and structural characterization of LytU
- Author
-
Tossavainen, H., primary, Raulinaitis, V., additional, and Permi, P., additional
- Published
- 2017
- Full Text
- View/download PDF
Catalog
3. Identification and structural characterization of LytU
- Author
-
Raulinaitis, V., primary, Tossavainen, H., additional, and Permi, P., additional
- Published
- 2017
- Full Text
- View/download PDF
4. Structural and Functional Insights Into Lysostaphin-Substrate Interaction.
- Author
-
Tossavainen H, Raulinaitis V, Kauppinen L, Pentikäinen U, Maaheimo H, and Permi P
- Abstract
Lysostaphin from Staphylococcus simulans and its family enzymes rapidly acquire prominence as the next generation agents in treatment of S. aureus infections. The specificity of lysostaphin is promoted by its C-terminal cell wall targeting domain selectivity toward pentaglycine bridges in S. aureus cell wall. Scission of these cross-links is carried out by its N-terminal catalytic domain, a zinc-dependent endopeptidase. Understanding the determinants affecting the efficiency of catalysis and strength and specificity of interactions lies at the heart of all lysostaphin family enzyme applications. To this end, we have used NMR, SAXS and molecular dynamics simulations to characterize lysostaphin structure and dynamics, to address the inter-domain interaction, the enzyme-substrate interaction as well as the catalytic properties of pentaglycine cleavage in solution. Our NMR structure confirms the recent crystal structure, yet, together with the molecular dynamics simulations, emphasizes the dynamic nature of the loops embracing the catalytic site. We found no evidence for inter-domain interaction, but, interestingly, the SAXS data delineate two preferred conformation subpopulations. Catalytic H329 and H360 were observed to bind a second zinc ion, which reduces lysostaphin pentaglycine cleaving activity. Binding of pentaglycine or its lysine derivatives to the targeting domain was found to be of very low affinity. The pentaglycine interaction site was located to the N-terminal groove of the domain. Notably, the targeting domain binds the peptidoglycan stem peptide Ala-d-γ-Glu-Lys-d-Ala-d-Ala with a much higher, micromolar affinity. Binding site mapping reveals two interaction sites of different affinities on the surface of the domain for this peptide. more...
- Published
- 2018
- Full Text
- View/download PDF
5. Identification and structural characterization of LytU, a unique peptidoglycan endopeptidase from the lysostaphin family.
- Author
-
Raulinaitis V, Tossavainen H, Aitio O, Juuti JT, Hiramatsu K, Kontinen V, and Permi P
- Subjects
- Amino Acid Sequence, Anti-Bacterial Agents chemistry, Binding Sites, Catalytic Domain, Cell Membrane chemistry, Cell Membrane metabolism, Endopeptidases genetics, Endopeptidases metabolism, Hydrogen-Ion Concentration, Lysostaphin metabolism, Mutation, Protein Binding, Protein Interaction Domains and Motifs, Proteolysis, Staphylococcus aureus enzymology, Staphylococcus aureus genetics, Staphylococcus aureus ultrastructure, Structure-Activity Relationship, Zinc metabolism, Endopeptidases chemistry, Lysostaphin chemistry
- Abstract
We introduce LytU, a short member of the lysostaphin family of zinc-dependent pentaglycine endopeptidases. It is a potential antimicrobial agent for S. aureus infections and its gene transcription is highly upregulated upon antibiotic treatments along with other genes involved in cell wall synthesis. We found this enzyme to be responsible for the opening of the cell wall peptidoglycan layer during cell divisions in S. aureus. LytU is anchored in the plasma membrane with the active part residing in the periplasmic space. It has a unique Ile/Lys insertion at position 151 that resides in the catalytic site-neighbouring loop and is vital for the enzymatic activity but not affecting the overall structure common to the lysostaphin family. Purified LytU lyses S. aureus cells and cleaves pentaglycine, a reaction conveniently monitored by NMR spectroscopy. Substituting the cofactor zinc ion with a copper or cobalt ion remarkably increases the rate of pentaglycine cleavage. NMR and isothermal titration calorimetry further reveal that, uniquely for its family, LytU is able to bind a second zinc ion which is coordinated by catalytic histidines and is therefore inhibitory. The pH-dependence and high affinity of binding carry further physiological implications. more...
- Published
- 2017
- Full Text
- View/download PDF
6. 1 H, 13 C and 15 N resonance assignments of the new lysostaphin family endopeptidase catalytic domain from Staphylococcus aureus.
- Author
-
Raulinaitis V, Tossavainen H, Aitio O, Seppala R, and Permi P
- Subjects
- Amino Acid Sequence, Lysostaphin metabolism, Catalytic Domain, Lysostaphin chemistry, Nuclear Magnetic Resonance, Biomolecular, Staphylococcus aureus enzymology
- Abstract
Lysostaphin family endopeptidases, produced by Staphylococcus genus, are zinc-dependent enzymes that cleave pentaglycine bridges of cell wall peptidoglycan. They act as autolysins to maintain cell wall metabolism or as toxins and weapons against competing strains. Consequently, these enzymes are compelling targets for new drugs as well as are potential antimicrobial agents themselves against Staphylococcus pathogens, which depend on cell wall to retain their immunity against antibiotics. The rapid spread of methicillin and vancomycin-resistant Staphylococcus aureus strains draws demand for new therapeutic approaches. S. aureus gene sa0205 was found to be implicated in resistance to vancomycin and synthesis of the bacteria cell wall. The gene encodes for a catalytic domain of a lysostaphin-type endopeptidase. We aim to obtain the structure of the Sa0205 catalytic domain, the first solution structure of the catalytic domain of the lysostaphin family enzymes. In addition, we are to investigate the apparent binding of the second zinc ion, which has not been previously reported for the enzyme group. Herein, we present the backbone and side chain resonance assignments of Sa0205 endopeptidase catalytic domain in its one and two zinc-bound forms. more...
- Published
- 2017
- Full Text
- View/download PDF
7. 1H, 13C and 15N resonance assignments of the major extracytoplasmic domain of the cell shape-determining protein MreC from Bacillus subtilis.
- Author
-
Kyburz A, Raulinaitis V, Koskela O, Kontinen V, Permi P, Kilpelainen I, and Seppala R
- Subjects
- Carbon Isotopes, Hydrogen, Nitrogen Isotopes, Protein Structure, Secondary, Protein Structure, Tertiary, Bacillus subtilis chemistry, Bacillus subtilis cytology, Bacterial Proteins chemistry, Cytoplasm chemistry, Nuclear Magnetic Resonance, Biomolecular
- Abstract
MreB, MreC and MreD are essential cell shape-determining morphogenetic proteins in Gram-positive and in Gram-negative bacteria. While MreB, the bacterial homologue of the eukaryotic cytoskeletal protein actin, has been extensively studied, the roles of MreC and MreD are less well understood. They both are transmembrane proteins. MreC has a predicted single transmembrane domain and the C-terminal part outside the cell membrane. MreC probably functions as a link between the intracellular cytoskeleton and the cell wall synthesizing machinery which is located at the outer surface of the cell membrane. Also proteins involved in cell wall synthesis participate in cell morphogenesis. How these two processes are coordinated is, however, poorly understood. Bacillus subtilis (BS), a non-pathogenic Gram-positive bacterium, is widely used as a model for Gram-positive pathogens, e.g. Staphylococcus aureus (SA). Currently, the structures of MreC from BS and SA are not known. As part of our efforts to elucidate the structure-function relationships of the morphogenetic protein complexes in Gram-positive bacteria, we present the backbone and side chain resonance assignments of the extracytoplasmic domain of MreC from BS. more...
- Published
- 2010
- Full Text
- View/download PDF
8. Segmental isotopic labeling of multi-domain and fusion proteins by protein trans-splicing in vivo and in vitro.
- Author
-
Muona M, Aranko AS, Raulinaitis V, and Iwaï H
- Subjects
- Base Sequence, DNA Polymerase III genetics, Escherichia coli genetics, Escherichia coli Proteins genetics, Genetic Vectors, In Vitro Techniques, Inteins, Nuclear Magnetic Resonance, Biomolecular, Plasmids genetics, Protein Structure, Tertiary, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Trans-Splicing, Isotope Labeling methods, Proteins chemistry, Proteins genetics
- Abstract
Segmental isotopic labeling is a powerful labeling technique for reducing nuclear magnetic resonance (NMR) signal overlap, which is associated with larger proteins by incorporating stable isotopes into only one region of a protein for NMR detections. Segmental isotopic labeling can not only reduce complexities of NMR spectra but also retain possibilities to carry out sequential resonance assignments by triple-resonance NMR experiments. We described in vivo (i.e., in Escherichia coli) and in vitro protocols for segmental isotopic labeling of multi-domain and fusion proteins via protein trans-splicing (PTS) using split DnaE intein without any refolding steps or alpha-thioester modification. The advantage of PTS approach is that it can be carried out in vivo by time-delayed dual-expression system with two controllable promoters. A segmentally isotope-labeled protein can be expressed in Escherichia coli within 1 d once required vectors are constructed. The total preparation time of a segmentally labeled sample can be as short as 7-13 d depending on the protocol used. more...
- Published
- 2010
- Full Text
- View/download PDF
9. Nicotine 5'-oxidation and methyl oxidation by P450 2A enzymes.
- Author
-
Murphy SE, Raulinaitis V, and Brown KM
- Subjects
- Chromatography, High Pressure Liquid, Cytochrome P-450 CYP2A6, Humans, In Vitro Techniques, Kinetics, Mixed Function Oxygenases metabolism, Nitrosamines, Smoking, Tritium, Aryl Hydrocarbon Hydroxylases metabolism, Cotinine metabolism, Microsomes, Liver metabolism, Nicotine analogs & derivatives, Nicotine metabolism, Steroid Hydroxylases metabolism
- Abstract
In smokers, the primary pathway of nicotine metabolism is P450 2A6-catalyzed 5'-oxidation. The nicotine Delta(5'(1'))-iminium ion product of this reaction is further metabolized to cotinine by aldehyde oxidase. Previous investigators have reported kinetic parameters for cotinine formation using human liver cytosol as a source of aldehyde oxidase. Using [5-(3)H]nicotine and radioflow high-performance liquid chromatography analysis, we determined kinetic parameters for nicotine 5'-oxidation by P450 2A6 and the closely related human extrahepatic P450 2A13 as well as the rodent P450s 2A3, 2A4, and 2A5. The formation of both cotinine and nicotine Delta(5'(1'))-iminium ion was monitored. The K(m) and V(max) values for P450 2A6 were 144 +/- 15 muM and 1.30 +/- 0.05 pmol/min/pmol, respectively. Previously reported K(m) values for cotinine formation by P450 2A6 in the presence of cytosol were much lower, ranging from 11 to 45 muM. P450 2A13 was a somewhat better catalyst of nicotine Delta(5'(1'))-iminium formation, with 2-fold lower K(m) and 2-fold higher V(max) values than P450 2A6. The rat P450 2A3 and the mouse P450 2A5, which are 85 and 84% identical to P450 2A6, were much more efficient catalysts of nicotine 5'-oxidation. P450 2A4 was not an efficient catalyst of nicotine metabolism. Whereas 5'-oxidation was the major pathway of nicotine metabolism for all five P450 2A enzymes, these enzymes also catalyzed methyl oxidation. Nornicotine, the product of this reaction was detected as 5 to 15% of the total nicotine metabolites. Nornicotine is the amine precursor to the esophageal carcinogen N'-nitrosonornicotine. Therefore, methyl oxidation of nicotine by P450 2A6 or P450 2A13 followed by nitrosation of nornicotine are possible endogenous pathways of N'-nitrosonornicotine formation. more...
- Published
- 2005
- Full Text
- View/download PDF
10. THERAPEUTIC DEMOCRACY: GROUP PROCESS AS A CORRECTIVE EMOTIONAL EXPERIENCE.
- Author
-
HOOVER KK, RAULINAITIS VB, and SPANER FE
- Subjects
- Humans, Democracy, Emotions, Group Processes, Interpersonal Relations, Motivation, Psychotherapy, Psychotherapy, Group, Schizophrenia
- Published
- 1965
- Full Text
- View/download PDF
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.