The Experts below are selected from a list of 210 Experts worldwide ranked by ideXlab platform
David Nicolas Chaperon - One of the best experts on this subject based on the ideXlab platform.
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Construction and complementation of in-frame deletions of the essential Escherichia coli thymidylate Kinase gene
Applied and Environmental Microbiology, 2006Co-Authors: David Nicolas ChaperonAbstract:This work reports the construction of Escherichia coli in-frame deletion strains of tmk, which encodes thymidylate Kinase, Tmk. The tmk gene is located at the third position of a putative five-gene operon at 24.9 min on the E. coli chromosome, which comprises the genes pabC, yceG, tmk, holB, and ycfH. To avoid potential polar effects on downstream genes of the operon, as well as recombination with plasmid-encoded tmk, the tmk gene was replaced by the Kanamycin resistance gene kka1, encoding amino glycoside 3'-phosphotransferase Kanamycin Kinase. The Kanamycin resistance gene is expressed under the control of the natural promoter(s) of the putative operon. The E. coli tmk gene is essential under any conditions tested. To show functional complementation in bacteria, the E. coli tmk gene was replaced by thymidylate Kinases of bacteriophage T4 gp1, E. coli tmk, Saccharomyces cerevisiae cdc8, or the Homo sapiens homologue, dTYMK. Growth of these transgenic E. coli strains is completely dependent on thymidylate Kinase activities of various origin expressed from plasmids. The substitution constructs show no polar effects on the downstream genes holB and ycfH with respect to cell viability. The presented transgenic bacteria could be of interest for testing of thymidylate Kinase-specific phosphorylation of nucleoside analogues that are used in therapies against cancer and infectious diseases.
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A method for the construction of in frame substitutions in operons: deletion of the essential Escherichia coli holB gene coding for a subunit of the DNA polymerase III holoenzyme.
Journal of microbiological methods, 2005Co-Authors: David Nicolas ChaperonAbstract:To investigate the putative five-gene operon at 24.9 min on the Escherichia coli genome, which comprises the genes pabC, yceG, tmk, holB and ycfH, a method for the construction of an in frame deletion strain of the essential E. coli holB gene was developed. HolB, also referred to as delta prime or yV, is a subunit of the DNA polymerase III (Pol III) holoenzyme. The holB gene was replaced by the Kanamycin resistance gene kka1, coding for amino glycoside 3V-phosphotransferase Kanamycin Kinase. The Kanamycin resistance gene was expressed under the control of the promoter(s) of the putative five-gene operon. The holB gene is essential for bacterial growth and the deletion of holB exhibits no polar effects on the adjacent genes tmk or ycfH in terms of cell viability. The method of the holB null construction presented in this work allows for a simplified studying of interactions between the different subunits of DNA polymerase III. D 2005 Elsevier B.V. All rights reserved.
Gerard D. Wright - One of the best experts on this subject based on the ideXlab platform.
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catalytic mechanism of enterococcal Kanamycin Kinase aph 3 iiia viscosity thio and solvent isotope effects support a theorell chance mechanism
Biochemistry, 1996Co-Authors: Geoffrey A Mckay, Gerard D. WrightAbstract:: Bacterial resistance to the aminoglycoside antibiotics is manifested primarily through the production of enzymes which covalently modify these drugs. The Enterococci and Staphylococci produce an ATP-dependent Kinase, APH(3')-IIIa, which phosphorylates such antibiotics as Kanamycin, amikacin, and neomycin, and this enzyme shows a Theorell-Chance kinetic mechanism by traditional product and analogue inhibitor analysis and by the alternative substrate diagnostic [McKay, G. A., & Wright, G. D. (1995) J. Biol. Chem. 270, 24686-24692]. We report that the APH(3')-IIIa exhibits small solvent (VH/VD approximately equal to 1.50) and thio effects (VATP/VATP gamma S = 2) indicating hydroxyl group deprotonation and nucleophilic attack on ATP do not significantly contribute to the overall steady-state rate. The enzymatic rates were determined with the viscogens PEG 8000, glycerol, and sucrose, and these experiments demonstrate that ATP binding and ADP release are diffusion controlled and that ADP release is solely rate limiting for APH(3')-IIIa. In addition, the slope of V/K for ATP vs relative viscosity is greater than the theoretical limit of 1, suggesting a possible enzyme conformational change upon binding of ATP. This new experimental evidence supports a Theorell-Chance mechanism for APH(3')-IIIa.
Geoffrey A Mckay - One of the best experts on this subject based on the ideXlab platform.
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catalytic mechanism of enterococcal Kanamycin Kinase aph 3 iiia viscosity thio and solvent isotope effects support a theorell chance mechanism
Biochemistry, 1996Co-Authors: Geoffrey A Mckay, Gerard D. WrightAbstract:: Bacterial resistance to the aminoglycoside antibiotics is manifested primarily through the production of enzymes which covalently modify these drugs. The Enterococci and Staphylococci produce an ATP-dependent Kinase, APH(3')-IIIa, which phosphorylates such antibiotics as Kanamycin, amikacin, and neomycin, and this enzyme shows a Theorell-Chance kinetic mechanism by traditional product and analogue inhibitor analysis and by the alternative substrate diagnostic [McKay, G. A., & Wright, G. D. (1995) J. Biol. Chem. 270, 24686-24692]. We report that the APH(3')-IIIa exhibits small solvent (VH/VD approximately equal to 1.50) and thio effects (VATP/VATP gamma S = 2) indicating hydroxyl group deprotonation and nucleophilic attack on ATP do not significantly contribute to the overall steady-state rate. The enzymatic rates were determined with the viscogens PEG 8000, glycerol, and sucrose, and these experiments demonstrate that ATP binding and ADP release are diffusion controlled and that ADP release is solely rate limiting for APH(3')-IIIa. In addition, the slope of V/K for ATP vs relative viscosity is greater than the theoretical limit of 1, suggesting a possible enzyme conformational change upon binding of ATP. This new experimental evidence supports a Theorell-Chance mechanism for APH(3')-IIIa.
Joseph Didonato - One of the best experts on this subject based on the ideXlab platform.
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Selection of an Eevolved Orthogonal Ne-Acetyllysine tRNAsynthetase that can Incorporate the Unnantural Amino Acid L-homocitrulline Site-specifically into Proteins
2014Co-Authors: Jaclyn Alatrash, Camelia Baleanu Gogonea, Stanly L. Hazen, Joseph DidonatoAbstract:Oxidatively damaged apolipoprotein A1 (apoA1, the main protein constituent of high density lipoprotein (HDL) the “good cholesterol”) has been isolated from circulating plasma and atherosclerosis plaque with modification of lysine amino acid residues to homocittruline (carbamyllysine) in apoA1 identified as a modification found increased in smokers that correlates with elevated cardiovascular risk for myocardial infarction (MI), stoke or death (1). This dysfunctional HDL molecule has been shown to be proinflammatory in nature and can activate the expression of cellular adhesion molecules in endothelial cells via NF-κB transcription factor activation (2). Here we designed ApoA1 to include specifically carbamylated lysine at predefined sites to further investigate the role of oxidative damage in atherosclerosis, leading to dysfunctional HDL. Current chemical and enzymatic oxidation of proteins (e.g. apoA1) results in incomplete and rarely site-specific oxidation of amino acids. Thus, genetic encoding of oxidized amino acids through orthogonal tRNA/aminoacyltRNA synthetase (RS) pairs offers the most reliable method for producing site specific oxidized proteins (3). We used an orthogonal tRNA/RS pair from Methanosarcina barkeri that has been evolved previously to incorporate the unnatural amino acid N-acetyllysine instead of pyrolysine (4) as a starting point to create a library of synthetase mutants that could utilize homocittruline (carbamyllysine) to specifically incorporate this uAA into proteins at defined sites. Using X-ray crystallographic information provided for the pyrolyisne tRNA synthetase (4) we replaced the pyrolysine with acetyllysine and observed which mutations were successful. Using this information and then replacing acetyllysine with carbamyllysine into the catalytic pocket we identified likely key residues that were within 6.5 angstroms of carbamyl lysine as it would sit in the catalytic pocket that might enhance hydrogen bonding or salt bridge electrostatic interactions between carbamyllysine and the catalytic pocket backbone amino acids. From the N-acetyllysine we identified A267(All), Leu270(All), Tyr 271(All), L274(All), F312(All) and C313(All). We used inverse PCR mutagenesis (4) to mutate each of these residues using two inverse PCR reactions and two mutagenic primers. A total of 6.4 x10 possible mutants could be made. After relegation of dilute isolated Bsa I restricted PCR plasmid DNA a library with a total of 1.5 x 10 was obtained. The library is in the process of being positively selected for incorporation of carbamyllysine into the Kanamycin Kinase gene at position 15 to suppress the encoded amber suppressor codon. Positive selected clones that grow on Kanamycin sulfate (150μg/ml) in the presence of 1mM carbamyllysine will be negatively selected against incorporation of any non-carbamyllysine amino acid in the absence of carbamyllysine using suppression of uracil phophoribosyl transferase at two amber codon sites (5 and 125) to incorporate 5-fluorouracil into cellular DNA, resulting in cell death. Clones which grow after being negatively selected for will then be positively selected a final time on kanaymcin sulfate plates in the presence of 1mM carbamyllysine for a second round of positive selection. Individual clones from this round of selection will be isolated and tested the clone encoding the synthase which best incorporates carbamyllysine at the lowest uAA concentration. The selected carbamyllysine tRNA synthetase will then be used to express a series of human apoA1 alleles containing amber codons in place of various lysine residues and assayed for its ability to form nascent HDL particles and to activate NF-κB and vascular adhesion molecule expression on endothelial cells.
V. N. Danilenko - One of the best experts on this subject based on the ideXlab platform.
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Identification of phosphorylation sites in aminoglycoside phosphotransferase VIII from Streptomyces rimosus
Biochemistry (Moscow), 2012Co-Authors: S. M. Elizarov, M. G. Alekseeva, F. N. Novikov, G. G. Chilov, D. A. Maslov, A. A. Shtil, V. N. DanilenkoAbstract:We demonstrate for the first time the role of phosphorylation in the regulation of activities of enzymes responsible for inactivation of aminoglycoside antibiotics. The aminoglycoside phosphotransferase VIII (APHVIII) from the actinobacterial strain Streptomyces rimosus ATCC 10970 is an enzyme regulated by protein Kinases. Two serine residues in APHVIII are shown to be phosphorylated by protein Kinases from extracts of the Kanamycin-resistant strain S. rimosus 683 (a derivative of strain ATCC 10970). Using site-directed mutagenesis and molecular modeling, we have identified the Ser146 residue in the activation loop of the enzyme as the key site for Ca^2+-dependent phosphorylation of APHVIII. Comparison of the Kanamycin Kinase activities of the unphosphorylated and phosphorylated forms of the initial and mutant APHVIII shows that the Ser146 modification leads to a 6–7-fold increase in the Kanamycin Kinase activity of APHVIII. Thus, Ser146 in the activation loop of APHVIII is crucial for the enzyme activity. The resistance of bacterial cells to Kanamycin increases proportionally. From the practical viewpoint, our results increase prospects for creation of highly effective test systems for selecting inhibitors of human and bacterial serine/threonine protein Kinases based on APHVIII constructs and corresponding human and bacterial serine/threonine protein Kinases.