The Experts below are selected from a list of 13338 Experts worldwide ranked by ideXlab platform
Juan Luis Asensio - One of the best experts on this subject based on the ideXlab platform.
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Multiple keys for a single lock: the unusual structural plasticity of the nucleotidyltransferase (4')/kanamycin complex.
Chemistry (Weinheim an der Bergstrasse Germany), 2012Co-Authors: Ruth Matesanz, José Fernando Díaz, Francisco Corzana, Andrés G. Santana, Agatha Bastida, Juan Luis AsensioAbstract:The most common mode of bacterial resistance to aminoglycoside antibiotics is the enzyme-catalysed chemical modification of the Drug. Over the last two decades, significant efforts in medicinal chemistry have been focused on the design of non- inactivable antibiotics. Unfortunately, this strategy has met with limited success on account of the remarkably wide substrate specificity of aminoglycoside-modifying enzymes. To understand the mechanisms behind substrate promiscuity, we have performed a comprehensive experimental and theoretical analysis of the molecular-recognition processes that lead to antibiotic Inactivation by Staphylococcus aureus nucleotidyltransferase 4'(ANT(4')), a clinically relevant protein. According to our results, the ability of this enzyme to inactivate structurally diverse polycationic molecules relies on three specific features of the catalytic region. First, the dominant role of electrostatics in aminoglycoside recognition, in combination with the significant extension of the enzyme anionic regions, confers to the protein/antibiotic complex a highly dynamic character. The motion deduced for the bound antibiotic seem to be essential for the enzyme action and probably provide a mechanism to explore alternative Drug Inactivation modes. Second, the nucleotide recognition is exclusively mediated by the inorganic fragment. In fact, even inorganic triphosphate can be employed as a substrate. Third, ANT(4') seems to be equipped with a duplicated basic catalyst that is able to promote Drug Inactivation through different reactive geometries. This particular combination of features explains the enzyme versatility and renders the design of non-inactivable derivatives a challenging task.
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nmr based analysis of aminoglycoside recognition by the resistance enzyme ant 4 the pattern of oh nh3 substitution determines the preferred antibiotic binding mode and is critical for Drug Inactivation
Journal of the American Chemical Society, 2008Co-Authors: Julia Revuelta, Francisco Corzana, Agatha Bastida, Tatiana Vacas, Mario Torrado, Carlos Gonzalez, Jesus Jimenezbarbero, Margarita Menendez, Juan Luis AsensioAbstract:The most significant mechanism of bacterial resistance to aminoglycosides is the enzymatic Inactivation of the Drug. Herein, we analyze several key aspects of the aminoglycoside recognition by the resistance enzyme ANT(4') from Staphylococcus aureus, employing NMR complemented with site-directed mutagenesis experiments and measurements of the enzymatic activity on newly synthesized kanamycin derivatives. From a methodological perspective, this analysis provides the first example reported for the use of transferred NOE (trNOE) experiments in the analysis of complex molecular recognition processes, characterized by the existence of simultaneous binding events of the ligand to different regions of a protein receptor. The obtained results show that, in favorable cases, these overlapping binding processes can be isolated employing site-directed mutagenesis and then independently analyzed. From a molecular recognition perspective, this work conclusively shows that the enzyme ANT(4') displays a wide tolerance to conformational variations in the Drug. Thus, according to the NMR data, kanamycin-A I/II linkage exhibits an unusual anti-Psi orientation in the ternary complex, which is in qualitative agreement with the previously reported crystallographic complex. In contrast, closely related, kanamycin-B is recognized by the enzyme in the syn-type arrangement for both glycosidic bonds. This observation together with the enzymatic activity displayed by ANT(4') against several synthetic kanamycin derivatives strongly suggests that the spatial distribution of positive charges within the aminoglycoside scaffold is the key feature that governs its preferred binding mode to the protein catalytic region and also the regioselectivity of the adenylation reaction. In contrast, the global shape of the antibiotic does not seem to be a critical factor. This feature represents a qualitative difference between the target A-site RNA and the resistance enzyme ANT(4') as aminoglycoside receptors.
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NMR-based analysis of aminoglycoside recognition by the resistance enzyme ANT(4'): the pattern of OH/NH3(+) substitution determines the preferred antibiotic binding mode and is critical for Drug Inactivation.
Journal of the American Chemical Society, 2008Co-Authors: Julia Revuelta, Francisco Corzana, Agatha Bastida, Tatiana Vacas, Mario Torrado, Carlos Gonzalez, Margarita Menendez, Jesús Jiménez-barbero, Juan Luis AsensioAbstract:The most significant mechanism of bacterial resistance to aminoglycosides is the enzymatic Inactivation of the Drug. Herein, we analyze several key aspects of the aminoglycoside recognition by the resistance enzyme ANT(4') from Staphylococcus aureus, employing NMR complemented with site-directed mutagenesis experiments and measurements of the enzymatic activity on newly synthesized kanamycin derivatives. From a methodological perspective, this analysis provides the first example reported for the use of transferred NOE (trNOE) experiments in the analysis of complex molecular recognition processes, characterized by the existence of simultaneous binding events of the ligand to different regions of a protein receptor. The obtained results show that, in favorable cases, these overlapping binding processes can be isolated employing site-directed mutagenesis and then independently analyzed. From a molecular recognition perspective, this work conclusively shows that the enzyme ANT(4') displays a wide tolerance to conformational variations in the Drug. Thus, according to the NMR data, kanamycin-A I/II linkage exhibits an unusual anti-Psi orientation in the ternary complex, which is in qualitative agreement with the previously reported crystallographic complex. In contrast, closely related, kanamycin-B is recognized by the enzyme in the syn-type arrangement for both glycosidic bonds. This observation together with the enzymatic activity displayed by ANT(4') against several synthetic kanamycin derivatives strongly suggests that the spatial distribution of positive charges within the aminoglycoside scaffold is the key feature that governs its preferred binding mode to the protein catalytic region and also the regioselectivity of the adenylation reaction. In contrast, the global shape of the antibiotic does not seem to be a critical factor. This feature represents a qualitative difference between the target A-site RNA and the resistance enzyme ANT(4') as aminoglycoside receptors.
Julia Revuelta - One of the best experts on this subject based on the ideXlab platform.
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nmr based analysis of aminoglycoside recognition by the resistance enzyme ant 4 the pattern of oh nh3 substitution determines the preferred antibiotic binding mode and is critical for Drug Inactivation
Journal of the American Chemical Society, 2008Co-Authors: Julia Revuelta, Francisco Corzana, Agatha Bastida, Tatiana Vacas, Mario Torrado, Carlos Gonzalez, Jesus Jimenezbarbero, Margarita Menendez, Juan Luis AsensioAbstract:The most significant mechanism of bacterial resistance to aminoglycosides is the enzymatic Inactivation of the Drug. Herein, we analyze several key aspects of the aminoglycoside recognition by the resistance enzyme ANT(4') from Staphylococcus aureus, employing NMR complemented with site-directed mutagenesis experiments and measurements of the enzymatic activity on newly synthesized kanamycin derivatives. From a methodological perspective, this analysis provides the first example reported for the use of transferred NOE (trNOE) experiments in the analysis of complex molecular recognition processes, characterized by the existence of simultaneous binding events of the ligand to different regions of a protein receptor. The obtained results show that, in favorable cases, these overlapping binding processes can be isolated employing site-directed mutagenesis and then independently analyzed. From a molecular recognition perspective, this work conclusively shows that the enzyme ANT(4') displays a wide tolerance to conformational variations in the Drug. Thus, according to the NMR data, kanamycin-A I/II linkage exhibits an unusual anti-Psi orientation in the ternary complex, which is in qualitative agreement with the previously reported crystallographic complex. In contrast, closely related, kanamycin-B is recognized by the enzyme in the syn-type arrangement for both glycosidic bonds. This observation together with the enzymatic activity displayed by ANT(4') against several synthetic kanamycin derivatives strongly suggests that the spatial distribution of positive charges within the aminoglycoside scaffold is the key feature that governs its preferred binding mode to the protein catalytic region and also the regioselectivity of the adenylation reaction. In contrast, the global shape of the antibiotic does not seem to be a critical factor. This feature represents a qualitative difference between the target A-site RNA and the resistance enzyme ANT(4') as aminoglycoside receptors.
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NMR-based analysis of aminoglycoside recognition by the resistance enzyme ANT(4'): the pattern of OH/NH3(+) substitution determines the preferred antibiotic binding mode and is critical for Drug Inactivation.
Journal of the American Chemical Society, 2008Co-Authors: Julia Revuelta, Francisco Corzana, Agatha Bastida, Tatiana Vacas, Mario Torrado, Carlos Gonzalez, Margarita Menendez, Jesús Jiménez-barbero, Juan Luis AsensioAbstract:The most significant mechanism of bacterial resistance to aminoglycosides is the enzymatic Inactivation of the Drug. Herein, we analyze several key aspects of the aminoglycoside recognition by the resistance enzyme ANT(4') from Staphylococcus aureus, employing NMR complemented with site-directed mutagenesis experiments and measurements of the enzymatic activity on newly synthesized kanamycin derivatives. From a methodological perspective, this analysis provides the first example reported for the use of transferred NOE (trNOE) experiments in the analysis of complex molecular recognition processes, characterized by the existence of simultaneous binding events of the ligand to different regions of a protein receptor. The obtained results show that, in favorable cases, these overlapping binding processes can be isolated employing site-directed mutagenesis and then independently analyzed. From a molecular recognition perspective, this work conclusively shows that the enzyme ANT(4') displays a wide tolerance to conformational variations in the Drug. Thus, according to the NMR data, kanamycin-A I/II linkage exhibits an unusual anti-Psi orientation in the ternary complex, which is in qualitative agreement with the previously reported crystallographic complex. In contrast, closely related, kanamycin-B is recognized by the enzyme in the syn-type arrangement for both glycosidic bonds. This observation together with the enzymatic activity displayed by ANT(4') against several synthetic kanamycin derivatives strongly suggests that the spatial distribution of positive charges within the aminoglycoside scaffold is the key feature that governs its preferred binding mode to the protein catalytic region and also the regioselectivity of the adenylation reaction. In contrast, the global shape of the antibiotic does not seem to be a critical factor. This feature represents a qualitative difference between the target A-site RNA and the resistance enzyme ANT(4') as aminoglycoside receptors.
Agatha Bastida - One of the best experts on this subject based on the ideXlab platform.
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Multiple keys for a single lock: the unusual structural plasticity of the nucleotidyltransferase (4')/kanamycin complex.
Chemistry (Weinheim an der Bergstrasse Germany), 2012Co-Authors: Ruth Matesanz, José Fernando Díaz, Francisco Corzana, Andrés G. Santana, Agatha Bastida, Juan Luis AsensioAbstract:The most common mode of bacterial resistance to aminoglycoside antibiotics is the enzyme-catalysed chemical modification of the Drug. Over the last two decades, significant efforts in medicinal chemistry have been focused on the design of non- inactivable antibiotics. Unfortunately, this strategy has met with limited success on account of the remarkably wide substrate specificity of aminoglycoside-modifying enzymes. To understand the mechanisms behind substrate promiscuity, we have performed a comprehensive experimental and theoretical analysis of the molecular-recognition processes that lead to antibiotic Inactivation by Staphylococcus aureus nucleotidyltransferase 4'(ANT(4')), a clinically relevant protein. According to our results, the ability of this enzyme to inactivate structurally diverse polycationic molecules relies on three specific features of the catalytic region. First, the dominant role of electrostatics in aminoglycoside recognition, in combination with the significant extension of the enzyme anionic regions, confers to the protein/antibiotic complex a highly dynamic character. The motion deduced for the bound antibiotic seem to be essential for the enzyme action and probably provide a mechanism to explore alternative Drug Inactivation modes. Second, the nucleotide recognition is exclusively mediated by the inorganic fragment. In fact, even inorganic triphosphate can be employed as a substrate. Third, ANT(4') seems to be equipped with a duplicated basic catalyst that is able to promote Drug Inactivation through different reactive geometries. This particular combination of features explains the enzyme versatility and renders the design of non-inactivable derivatives a challenging task.
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nmr based analysis of aminoglycoside recognition by the resistance enzyme ant 4 the pattern of oh nh3 substitution determines the preferred antibiotic binding mode and is critical for Drug Inactivation
Journal of the American Chemical Society, 2008Co-Authors: Julia Revuelta, Francisco Corzana, Agatha Bastida, Tatiana Vacas, Mario Torrado, Carlos Gonzalez, Jesus Jimenezbarbero, Margarita Menendez, Juan Luis AsensioAbstract:The most significant mechanism of bacterial resistance to aminoglycosides is the enzymatic Inactivation of the Drug. Herein, we analyze several key aspects of the aminoglycoside recognition by the resistance enzyme ANT(4') from Staphylococcus aureus, employing NMR complemented with site-directed mutagenesis experiments and measurements of the enzymatic activity on newly synthesized kanamycin derivatives. From a methodological perspective, this analysis provides the first example reported for the use of transferred NOE (trNOE) experiments in the analysis of complex molecular recognition processes, characterized by the existence of simultaneous binding events of the ligand to different regions of a protein receptor. The obtained results show that, in favorable cases, these overlapping binding processes can be isolated employing site-directed mutagenesis and then independently analyzed. From a molecular recognition perspective, this work conclusively shows that the enzyme ANT(4') displays a wide tolerance to conformational variations in the Drug. Thus, according to the NMR data, kanamycin-A I/II linkage exhibits an unusual anti-Psi orientation in the ternary complex, which is in qualitative agreement with the previously reported crystallographic complex. In contrast, closely related, kanamycin-B is recognized by the enzyme in the syn-type arrangement for both glycosidic bonds. This observation together with the enzymatic activity displayed by ANT(4') against several synthetic kanamycin derivatives strongly suggests that the spatial distribution of positive charges within the aminoglycoside scaffold is the key feature that governs its preferred binding mode to the protein catalytic region and also the regioselectivity of the adenylation reaction. In contrast, the global shape of the antibiotic does not seem to be a critical factor. This feature represents a qualitative difference between the target A-site RNA and the resistance enzyme ANT(4') as aminoglycoside receptors.
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NMR-based analysis of aminoglycoside recognition by the resistance enzyme ANT(4'): the pattern of OH/NH3(+) substitution determines the preferred antibiotic binding mode and is critical for Drug Inactivation.
Journal of the American Chemical Society, 2008Co-Authors: Julia Revuelta, Francisco Corzana, Agatha Bastida, Tatiana Vacas, Mario Torrado, Carlos Gonzalez, Margarita Menendez, Jesús Jiménez-barbero, Juan Luis AsensioAbstract:The most significant mechanism of bacterial resistance to aminoglycosides is the enzymatic Inactivation of the Drug. Herein, we analyze several key aspects of the aminoglycoside recognition by the resistance enzyme ANT(4') from Staphylococcus aureus, employing NMR complemented with site-directed mutagenesis experiments and measurements of the enzymatic activity on newly synthesized kanamycin derivatives. From a methodological perspective, this analysis provides the first example reported for the use of transferred NOE (trNOE) experiments in the analysis of complex molecular recognition processes, characterized by the existence of simultaneous binding events of the ligand to different regions of a protein receptor. The obtained results show that, in favorable cases, these overlapping binding processes can be isolated employing site-directed mutagenesis and then independently analyzed. From a molecular recognition perspective, this work conclusively shows that the enzyme ANT(4') displays a wide tolerance to conformational variations in the Drug. Thus, according to the NMR data, kanamycin-A I/II linkage exhibits an unusual anti-Psi orientation in the ternary complex, which is in qualitative agreement with the previously reported crystallographic complex. In contrast, closely related, kanamycin-B is recognized by the enzyme in the syn-type arrangement for both glycosidic bonds. This observation together with the enzymatic activity displayed by ANT(4') against several synthetic kanamycin derivatives strongly suggests that the spatial distribution of positive charges within the aminoglycoside scaffold is the key feature that governs its preferred binding mode to the protein catalytic region and also the regioselectivity of the adenylation reaction. In contrast, the global shape of the antibiotic does not seem to be a critical factor. This feature represents a qualitative difference between the target A-site RNA and the resistance enzyme ANT(4') as aminoglycoside receptors.
Francisco Corzana - One of the best experts on this subject based on the ideXlab platform.
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Multiple keys for a single lock: the unusual structural plasticity of the nucleotidyltransferase (4')/kanamycin complex.
Chemistry (Weinheim an der Bergstrasse Germany), 2012Co-Authors: Ruth Matesanz, José Fernando Díaz, Francisco Corzana, Andrés G. Santana, Agatha Bastida, Juan Luis AsensioAbstract:The most common mode of bacterial resistance to aminoglycoside antibiotics is the enzyme-catalysed chemical modification of the Drug. Over the last two decades, significant efforts in medicinal chemistry have been focused on the design of non- inactivable antibiotics. Unfortunately, this strategy has met with limited success on account of the remarkably wide substrate specificity of aminoglycoside-modifying enzymes. To understand the mechanisms behind substrate promiscuity, we have performed a comprehensive experimental and theoretical analysis of the molecular-recognition processes that lead to antibiotic Inactivation by Staphylococcus aureus nucleotidyltransferase 4'(ANT(4')), a clinically relevant protein. According to our results, the ability of this enzyme to inactivate structurally diverse polycationic molecules relies on three specific features of the catalytic region. First, the dominant role of electrostatics in aminoglycoside recognition, in combination with the significant extension of the enzyme anionic regions, confers to the protein/antibiotic complex a highly dynamic character. The motion deduced for the bound antibiotic seem to be essential for the enzyme action and probably provide a mechanism to explore alternative Drug Inactivation modes. Second, the nucleotide recognition is exclusively mediated by the inorganic fragment. In fact, even inorganic triphosphate can be employed as a substrate. Third, ANT(4') seems to be equipped with a duplicated basic catalyst that is able to promote Drug Inactivation through different reactive geometries. This particular combination of features explains the enzyme versatility and renders the design of non-inactivable derivatives a challenging task.
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nmr based analysis of aminoglycoside recognition by the resistance enzyme ant 4 the pattern of oh nh3 substitution determines the preferred antibiotic binding mode and is critical for Drug Inactivation
Journal of the American Chemical Society, 2008Co-Authors: Julia Revuelta, Francisco Corzana, Agatha Bastida, Tatiana Vacas, Mario Torrado, Carlos Gonzalez, Jesus Jimenezbarbero, Margarita Menendez, Juan Luis AsensioAbstract:The most significant mechanism of bacterial resistance to aminoglycosides is the enzymatic Inactivation of the Drug. Herein, we analyze several key aspects of the aminoglycoside recognition by the resistance enzyme ANT(4') from Staphylococcus aureus, employing NMR complemented with site-directed mutagenesis experiments and measurements of the enzymatic activity on newly synthesized kanamycin derivatives. From a methodological perspective, this analysis provides the first example reported for the use of transferred NOE (trNOE) experiments in the analysis of complex molecular recognition processes, characterized by the existence of simultaneous binding events of the ligand to different regions of a protein receptor. The obtained results show that, in favorable cases, these overlapping binding processes can be isolated employing site-directed mutagenesis and then independently analyzed. From a molecular recognition perspective, this work conclusively shows that the enzyme ANT(4') displays a wide tolerance to conformational variations in the Drug. Thus, according to the NMR data, kanamycin-A I/II linkage exhibits an unusual anti-Psi orientation in the ternary complex, which is in qualitative agreement with the previously reported crystallographic complex. In contrast, closely related, kanamycin-B is recognized by the enzyme in the syn-type arrangement for both glycosidic bonds. This observation together with the enzymatic activity displayed by ANT(4') against several synthetic kanamycin derivatives strongly suggests that the spatial distribution of positive charges within the aminoglycoside scaffold is the key feature that governs its preferred binding mode to the protein catalytic region and also the regioselectivity of the adenylation reaction. In contrast, the global shape of the antibiotic does not seem to be a critical factor. This feature represents a qualitative difference between the target A-site RNA and the resistance enzyme ANT(4') as aminoglycoside receptors.
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NMR-based analysis of aminoglycoside recognition by the resistance enzyme ANT(4'): the pattern of OH/NH3(+) substitution determines the preferred antibiotic binding mode and is critical for Drug Inactivation.
Journal of the American Chemical Society, 2008Co-Authors: Julia Revuelta, Francisco Corzana, Agatha Bastida, Tatiana Vacas, Mario Torrado, Carlos Gonzalez, Margarita Menendez, Jesús Jiménez-barbero, Juan Luis AsensioAbstract:The most significant mechanism of bacterial resistance to aminoglycosides is the enzymatic Inactivation of the Drug. Herein, we analyze several key aspects of the aminoglycoside recognition by the resistance enzyme ANT(4') from Staphylococcus aureus, employing NMR complemented with site-directed mutagenesis experiments and measurements of the enzymatic activity on newly synthesized kanamycin derivatives. From a methodological perspective, this analysis provides the first example reported for the use of transferred NOE (trNOE) experiments in the analysis of complex molecular recognition processes, characterized by the existence of simultaneous binding events of the ligand to different regions of a protein receptor. The obtained results show that, in favorable cases, these overlapping binding processes can be isolated employing site-directed mutagenesis and then independently analyzed. From a molecular recognition perspective, this work conclusively shows that the enzyme ANT(4') displays a wide tolerance to conformational variations in the Drug. Thus, according to the NMR data, kanamycin-A I/II linkage exhibits an unusual anti-Psi orientation in the ternary complex, which is in qualitative agreement with the previously reported crystallographic complex. In contrast, closely related, kanamycin-B is recognized by the enzyme in the syn-type arrangement for both glycosidic bonds. This observation together with the enzymatic activity displayed by ANT(4') against several synthetic kanamycin derivatives strongly suggests that the spatial distribution of positive charges within the aminoglycoside scaffold is the key feature that governs its preferred binding mode to the protein catalytic region and also the regioselectivity of the adenylation reaction. In contrast, the global shape of the antibiotic does not seem to be a critical factor. This feature represents a qualitative difference between the target A-site RNA and the resistance enzyme ANT(4') as aminoglycoside receptors.
Tatiana Vacas - One of the best experts on this subject based on the ideXlab platform.
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nmr based analysis of aminoglycoside recognition by the resistance enzyme ant 4 the pattern of oh nh3 substitution determines the preferred antibiotic binding mode and is critical for Drug Inactivation
Journal of the American Chemical Society, 2008Co-Authors: Julia Revuelta, Francisco Corzana, Agatha Bastida, Tatiana Vacas, Mario Torrado, Carlos Gonzalez, Jesus Jimenezbarbero, Margarita Menendez, Juan Luis AsensioAbstract:The most significant mechanism of bacterial resistance to aminoglycosides is the enzymatic Inactivation of the Drug. Herein, we analyze several key aspects of the aminoglycoside recognition by the resistance enzyme ANT(4') from Staphylococcus aureus, employing NMR complemented with site-directed mutagenesis experiments and measurements of the enzymatic activity on newly synthesized kanamycin derivatives. From a methodological perspective, this analysis provides the first example reported for the use of transferred NOE (trNOE) experiments in the analysis of complex molecular recognition processes, characterized by the existence of simultaneous binding events of the ligand to different regions of a protein receptor. The obtained results show that, in favorable cases, these overlapping binding processes can be isolated employing site-directed mutagenesis and then independently analyzed. From a molecular recognition perspective, this work conclusively shows that the enzyme ANT(4') displays a wide tolerance to conformational variations in the Drug. Thus, according to the NMR data, kanamycin-A I/II linkage exhibits an unusual anti-Psi orientation in the ternary complex, which is in qualitative agreement with the previously reported crystallographic complex. In contrast, closely related, kanamycin-B is recognized by the enzyme in the syn-type arrangement for both glycosidic bonds. This observation together with the enzymatic activity displayed by ANT(4') against several synthetic kanamycin derivatives strongly suggests that the spatial distribution of positive charges within the aminoglycoside scaffold is the key feature that governs its preferred binding mode to the protein catalytic region and also the regioselectivity of the adenylation reaction. In contrast, the global shape of the antibiotic does not seem to be a critical factor. This feature represents a qualitative difference between the target A-site RNA and the resistance enzyme ANT(4') as aminoglycoside receptors.
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NMR-based analysis of aminoglycoside recognition by the resistance enzyme ANT(4'): the pattern of OH/NH3(+) substitution determines the preferred antibiotic binding mode and is critical for Drug Inactivation.
Journal of the American Chemical Society, 2008Co-Authors: Julia Revuelta, Francisco Corzana, Agatha Bastida, Tatiana Vacas, Mario Torrado, Carlos Gonzalez, Margarita Menendez, Jesús Jiménez-barbero, Juan Luis AsensioAbstract:The most significant mechanism of bacterial resistance to aminoglycosides is the enzymatic Inactivation of the Drug. Herein, we analyze several key aspects of the aminoglycoside recognition by the resistance enzyme ANT(4') from Staphylococcus aureus, employing NMR complemented with site-directed mutagenesis experiments and measurements of the enzymatic activity on newly synthesized kanamycin derivatives. From a methodological perspective, this analysis provides the first example reported for the use of transferred NOE (trNOE) experiments in the analysis of complex molecular recognition processes, characterized by the existence of simultaneous binding events of the ligand to different regions of a protein receptor. The obtained results show that, in favorable cases, these overlapping binding processes can be isolated employing site-directed mutagenesis and then independently analyzed. From a molecular recognition perspective, this work conclusively shows that the enzyme ANT(4') displays a wide tolerance to conformational variations in the Drug. Thus, according to the NMR data, kanamycin-A I/II linkage exhibits an unusual anti-Psi orientation in the ternary complex, which is in qualitative agreement with the previously reported crystallographic complex. In contrast, closely related, kanamycin-B is recognized by the enzyme in the syn-type arrangement for both glycosidic bonds. This observation together with the enzymatic activity displayed by ANT(4') against several synthetic kanamycin derivatives strongly suggests that the spatial distribution of positive charges within the aminoglycoside scaffold is the key feature that governs its preferred binding mode to the protein catalytic region and also the regioselectivity of the adenylation reaction. In contrast, the global shape of the antibiotic does not seem to be a critical factor. This feature represents a qualitative difference between the target A-site RNA and the resistance enzyme ANT(4') as aminoglycoside receptors.
