The Experts below are selected from a list of 210 Experts worldwide ranked by ideXlab platform
James R. Knox - One of the best experts on this subject based on the ideXlab platform.
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Enzymes of vancomycin resistance: The structure of D-Alanine-D-lactate ligase of naturally resistant Leuconostoc mesenteroides
Structure, 2000Co-Authors: Alexandre P. Kuzin, Jodi Jorczak-baillass, Vicki L. Healy, Tao Sun, Christopher T. Walsh, James R. KnoxAbstract:Background: The bacterial cell wall and the enzymes that synthesize it are targets of glycopeptide antibiotics (vancomycins and teicoplanins) and β-lactams (penicillins and cephalosporins). Biosynthesis of cell wall peptidoglycan requires a crosslinking of peptidyl moieties on adjacent glycan strands. The D-Alanine-D-Alanine transpeptidase, which catalyzes this crosslinking, is the target of β-lactam antibiotics. Glycopeptides, in contrast, do not inhibit an enzyme, but bind directly to D-Alanine-D-Alanine and prevent subsequent crosslinking by the transpeptidase. Clinical resistance to vancomycin in enterococcal pathogens has been traced to altered ligases producing D-Alanine-D-lactate rather than D-Alanine-D-Alanine. Results: The structure of a D-Alanine-D-lactate ligase has been determined by multiple anomalous dispersion (MAD) phasing to 2.4 Å resolution. Co- crystallization of the Leuconostoc mesenteroides LmDdl2 ligase with ATP and a di-D-methylphosphinate produced ADP and a phosphinophosphate analog of the reaction intermediate of cell wall peptidoglycan biosynthesis. Comparison of this D-Alanine-D-lactate ligase with the known structure of DdlB D-Alanine-D- Alanine ligase, a wild-type enzyme that does not provide vancomycin resistance, reveals alterations in the size and hydrophobicity of the site for D-lactate binding (subsite 2). A decrease was noted in the ability of the ligase to hydrogen bond a substrate molecule entering subsite 2. Conclusions: Structural differences at subsite 2 of the D-Alanine-D-lactate ligase help explain a substrate specificity shift (D-Alanine to D-lactate) leading to remodeled cell wall peptidoglycan and vancomycin resistance in Gram-positive pathogens.
Jean-michel Masson - One of the best experts on this subject based on the ideXlab platform.
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Alanine-stretch scanning mutagenesis: a simple and efficient method to probe protein structure and function
Nucleic Acids Research, 1997Co-Authors: Fabrice Lefevre, Marie Hélène Rémy, Jean-michel MassonAbstract:We have developed a foolproof method to substitute a stretch of residues by Alanines. After the introduction of a PstI site by IPCR, thus creating two Alanine codons, additional codons are introduced at this site through the use of an ‘Alanine-stretch cartridge’. These cartridges comprise an antibiotic resistance gene flanked on both sides by Alanine codons. Excision of the resistance gene by PvuII then yields the correct insertion of codons. The method is both highly reliable and flexible and should be of general use.
Alexandre P. Kuzin - One of the best experts on this subject based on the ideXlab platform.
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Enzymes of vancomycin resistance: The structure of D-Alanine-D-lactate ligase of naturally resistant Leuconostoc mesenteroides
Structure, 2000Co-Authors: Alexandre P. Kuzin, Jodi Jorczak-baillass, Vicki L. Healy, Tao Sun, Christopher T. Walsh, James R. KnoxAbstract:Background: The bacterial cell wall and the enzymes that synthesize it are targets of glycopeptide antibiotics (vancomycins and teicoplanins) and β-lactams (penicillins and cephalosporins). Biosynthesis of cell wall peptidoglycan requires a crosslinking of peptidyl moieties on adjacent glycan strands. The D-Alanine-D-Alanine transpeptidase, which catalyzes this crosslinking, is the target of β-lactam antibiotics. Glycopeptides, in contrast, do not inhibit an enzyme, but bind directly to D-Alanine-D-Alanine and prevent subsequent crosslinking by the transpeptidase. Clinical resistance to vancomycin in enterococcal pathogens has been traced to altered ligases producing D-Alanine-D-lactate rather than D-Alanine-D-Alanine. Results: The structure of a D-Alanine-D-lactate ligase has been determined by multiple anomalous dispersion (MAD) phasing to 2.4 Å resolution. Co- crystallization of the Leuconostoc mesenteroides LmDdl2 ligase with ATP and a di-D-methylphosphinate produced ADP and a phosphinophosphate analog of the reaction intermediate of cell wall peptidoglycan biosynthesis. Comparison of this D-Alanine-D-lactate ligase with the known structure of DdlB D-Alanine-D- Alanine ligase, a wild-type enzyme that does not provide vancomycin resistance, reveals alterations in the size and hydrophobicity of the site for D-lactate binding (subsite 2). A decrease was noted in the ability of the ligase to hydrogen bond a substrate molecule entering subsite 2. Conclusions: Structural differences at subsite 2 of the D-Alanine-D-lactate ligase help explain a substrate specificity shift (D-Alanine to D-lactate) leading to remodeled cell wall peptidoglycan and vancomycin resistance in Gram-positive pathogens.
Fabrice Lefevre - One of the best experts on this subject based on the ideXlab platform.
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Alanine-stretch scanning mutagenesis: a simple and efficient method to probe protein structure and function
Nucleic Acids Research, 1997Co-Authors: Fabrice Lefevre, Marie Hélène Rémy, Jean-michel MassonAbstract:We have developed a foolproof method to substitute a stretch of residues by Alanines. After the introduction of a PstI site by IPCR, thus creating two Alanine codons, additional codons are introduced at this site through the use of an ‘Alanine-stretch cartridge’. These cartridges comprise an antibiotic resistance gene flanked on both sides by Alanine codons. Excision of the resistance gene by PvuII then yields the correct insertion of codons. The method is both highly reliable and flexible and should be of general use.
Angel F Lopez - One of the best experts on this subject based on the ideXlab platform.
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a single tyrosine residue in the membrane proximal domain of the granulocyte macrophage colony stimulating factor interleukin il 3 and il 5 receptor common β chain is necessary and sufficient for high affinity binding and signaling by all three ligan
Journal of Biological Chemistry, 1996Co-Authors: Joanna M. Woodcock, B Zacharakis, Christopher J Bagley, Angel F LopezAbstract:Abstract The β-chain of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5) receptors functions as a communal receptor subunit and is often referred to as β common (βc). Analogous to other shared receptor subunits including gp130 and the IL-2R γ chain, βc mediates high affinity binding and signal transduction of all of its ligands. It is not clear, however, how these common receptor subunits can recognize several ligands and indeed whether they exhibit a common binding pocket to accomplish this. We have performed molecular modeling of βc based on the known structures of the growth hormone and prolactin receptors and targeted the putative F′-G′ loop for mutagenesis. Substitution of this whole predicted loop region with Alanines completely abrogated high affinity binding of GM-CSF, IL-3, and IL-5. Individual Alanine substitutions across the loop revealed that a single residue, Tyr421, is critical for high affinity binding of GM-CSF, IL-3, and IL-5, whereas Alanine substitution of adjacent residues has little or no effect on high affinity binding. Significantly, reintroducing Tyr421 into the polyAlanine-substituted mutant restored high affinity ligand binding of GM-CSF, IL-3, and IL-5, indicating that within this region the tyrosine residue alone is sufficient for high affinity ligand interaction. Functional studies measuring STAT5 activation revealed that Alanine substitution of Tyr421 severely impaired the ability of βc to signal. These results show for the first time that a single residue in a shared receptor subunit acts as a binding determinant for different ligands and may have implications for other receptor systems where communal receptor subunits exhibit hydrophobic residues in their putative F′-G′ loops. These results also raise the possibility that a single compound targeted to this region may simultaneously inhibit the binding and function of multiple cytokines.
