The Experts below are selected from a list of 168 Experts worldwide ranked by ideXlab platform
Jiří Damborský - One of the best experts on this subject based on the ideXlab platform.
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modification of activity and specificity of haloalkane dehalogenase from sphingomonas paucimobilis ut26 by engineering of its entrance tunnel
Journal of Biological Chemistry, 2003Co-Authors: Radka Chaloupkova, Jana Sýkorova, Marta Monincova, Martina Pavlová, Andrea Jesenska, Yuji Nagata, Masataka Tsuda, Zbynek Prokop, Jiří DamborskýAbstract:Abstract Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and Nonpolar Amino Acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring Amino Acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.
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modification of activity and specificity of haloalkane dehalogenase from sphingomonas paucimobilis ut26 by engineering of its entrance tunnel
Journal of Biological Chemistry, 2003Co-Authors: Radka Chaloupkova, Jana Sýkorova, Marta Monincova, Martina Pavlová, Andrea Jesenska, Yuji Nagata, Masataka Tsuda, Zbyňek Prokop, Jiří DamborskýAbstract:Abstract Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and Nonpolar Amino Acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring Amino Acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.
Radka Chaloupkova - One of the best experts on this subject based on the ideXlab platform.
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modification of activity and specificity of haloalkane dehalogenase from sphingomonas paucimobilis ut26 by engineering of its entrance tunnel
Journal of Biological Chemistry, 2003Co-Authors: Radka Chaloupkova, Jana Sýkorova, Marta Monincova, Martina Pavlová, Andrea Jesenska, Yuji Nagata, Masataka Tsuda, Zbynek Prokop, Jiří DamborskýAbstract:Abstract Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and Nonpolar Amino Acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring Amino Acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.
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modification of activity and specificity of haloalkane dehalogenase from sphingomonas paucimobilis ut26 by engineering of its entrance tunnel
Journal of Biological Chemistry, 2003Co-Authors: Radka Chaloupkova, Jana Sýkorova, Marta Monincova, Martina Pavlová, Andrea Jesenska, Yuji Nagata, Masataka Tsuda, Zbyňek Prokop, Jiří DamborskýAbstract:Abstract Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and Nonpolar Amino Acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring Amino Acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.
J B Reiser - One of the best experts on this subject based on the ideXlab platform.
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structural insights into the substrate recognition and reaction specificity of the plp dependent fold type i isoleucine 2 epimerase from lactobacillus buchneri
Biochimie, 2017Co-Authors: Rida Awad, Pierre Gans, J B ReiserAbstract:The isoleucine 2-epimerase from Lactobacillus buchneri has been previously identified and characterized to catalyze the pyridoxal 5'-phosphate (PLP)-dependent racemization and epimerization of a broad spectrum of Nonpolar Amino Acids from L- to D-form and vice versa, in particular isoleucine. In this study, crystal structures of both native and PLP-complex forms of this racemase are presented at 2.6 and 2.15 A resolution, respectively. Both structures show that the protein belongs to the fold-type I subgroup of PLP-dependent enzymes and is very close to Aminobutyrate Aminotransferases family, as it has been suspected because of their sequence homology. The extensive structural comparison with fold-type I enzymes with known Amino Acid racemization activities, including the α-Amino-e-caprolactam racemase from Achromobacter obae and the cystathionine β-lyase from Escherichia coli, allows us to identify the active site residues responsible for its Nonpolar Amino Acid recognition and reactivity specificity. Our observations also suggest that the racemization reaction by the fold-type I racemases may generally occur thanks to a revised two-base mechanism. Lastly, both structures reveal details on the conformational changes provoked by PLP binding that suggest an induced fit of the active site "entrance door", necessary to accommodate PLP and substrate molecules.
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Structural insights into the substrate recognition and reaction specificity of the PLP-dependent fold-type I isoleucine 2-epimerase from Lactobacillus buchneri.
Biochimie, 2017Co-Authors: Rida Awad, Pierre Gans, J B ReiserAbstract:The isoleucine 2-epimerase from Lactobacillus buchneri has been previously identified and characterized to catalyze the pyridoxal 5'-phosphate (PLP)-dependent racemization and epimerization of a broad spectrum of Nonpolar Amino Acids from L- to D-form and vice versa, in particular isoleucine. In this study, crystal structures of both native and PLP-complex forms of this racemase are presented at 2.6 and 2.15 Å resolution, respectively. Both structures show that the protein belongs to the fold-type I subgroup of PLP-dependent enzymes and is very close to Aminobutyrate Aminotransferases family, as it has been suspected because of their sequence homology. The extensive structural comparison with fold-type I enzymes with known Amino Acid racemization activities, including the α-Amino-ε-caprolactam racemase from Achromobacter obae and the cystathionine β-lyase from Escherichia coli, allows us to identify the active site residues responsible for its Nonpolar Amino Acid recognition and reactivity specificity. Our observations also suggest that the racemization reaction by the fold-type I racemases may generally occur thanks to a revised two-base mechanism. Lastly, both structures reveal details on the conformational changes provoked by PLP binding that suggest an induced fit of the active site "entrance door", necessary to accommodate PLP and substrate molecules.
Jana Sýkorova - One of the best experts on this subject based on the ideXlab platform.
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modification of activity and specificity of haloalkane dehalogenase from sphingomonas paucimobilis ut26 by engineering of its entrance tunnel
Journal of Biological Chemistry, 2003Co-Authors: Radka Chaloupkova, Jana Sýkorova, Marta Monincova, Martina Pavlová, Andrea Jesenska, Yuji Nagata, Masataka Tsuda, Zbynek Prokop, Jiří DamborskýAbstract:Abstract Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and Nonpolar Amino Acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring Amino Acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.
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modification of activity and specificity of haloalkane dehalogenase from sphingomonas paucimobilis ut26 by engineering of its entrance tunnel
Journal of Biological Chemistry, 2003Co-Authors: Radka Chaloupkova, Jana Sýkorova, Marta Monincova, Martina Pavlová, Andrea Jesenska, Yuji Nagata, Masataka Tsuda, Zbyňek Prokop, Jiří DamborskýAbstract:Abstract Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and Nonpolar Amino Acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring Amino Acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.
Marta Monincova - One of the best experts on this subject based on the ideXlab platform.
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modification of activity and specificity of haloalkane dehalogenase from sphingomonas paucimobilis ut26 by engineering of its entrance tunnel
Journal of Biological Chemistry, 2003Co-Authors: Radka Chaloupkova, Jana Sýkorova, Marta Monincova, Martina Pavlová, Andrea Jesenska, Yuji Nagata, Masataka Tsuda, Zbynek Prokop, Jiří DamborskýAbstract:Abstract Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and Nonpolar Amino Acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring Amino Acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.
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modification of activity and specificity of haloalkane dehalogenase from sphingomonas paucimobilis ut26 by engineering of its entrance tunnel
Journal of Biological Chemistry, 2003Co-Authors: Radka Chaloupkova, Jana Sýkorova, Marta Monincova, Martina Pavlová, Andrea Jesenska, Yuji Nagata, Masataka Tsuda, Zbyňek Prokop, Jiří DamborskýAbstract:Abstract Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and Nonpolar Amino Acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring Amino Acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.
