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Samir Bejar - One of the best experts on this subject based on the ideXlab platform.
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rational design of bacillus stearothermophilus us100 l arabinose isomerase potential applications for d tagatose production
Biochimie, 2009Co-Authors: Moez Rhimi, Richard Haser, Nushin Aghajari, Emmanuelle Maguin, Hichem Chouayekh, Samir BejarAbstract:Abstract l -Arabinose Isomerases catalyze the bioconversion of d -galactose into d -tagatose. With the aim of producing an enzyme optimized for d -tagatose production, three Bacillus stearothermophilus US100 l -arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 °C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0–7.0 and an optimal activity around 50–65 °C, temperatures at which the enzyme was stable without addition of metal ions.
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Rational design of Bacillus stearothermophilus US100 L-arabinose isomerase: potential applications for D-tagatose production
Biochimie, 2009Co-Authors: Moez Rhimi, Richard Haser, Nushin Aghajari, Emmanuelle Maguin, Hichem Chouayekh, Michel Juy, Samir BejarAbstract:L-arabinose Isomerases catalyze the bioconversion of D-galactose into D-tagatose. With the aim of producing an enzyme optimized for D-tagatose production, three Bacillus stearothermophilus US100 L-arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 degrees C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0-7.0 and an optimal activity around 50-65 degrees C, temperatures at which the enzyme was stable without addition of metal ions.
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probing the essential catalytic residues and substrate affinity in the thermoactive bacillus stearothermophilus us100 l arabinose isomerase by site directed mutagenesis
Journal of Bacteriology, 2007Co-Authors: Moez Rhimi, Richard Haser, Nushin Aghajari, Samir BejarAbstract:The l-arabinose isomerase (l-AI) from Bacillus stearothermophilus US100 is characterized by its high thermoactivity and catalytic efficiency. Furthermore, as opposed to the majority of l-arabinose Isomerases, this enzyme requires metallic ions for its thermostability rather than for its activity. These features make US100 l-AI attractive as a template for industrial use. Based on previously solved crystal structures and sequence alignments, we identified amino acids that are putatively important for the US100 l-AI isomerization reaction. Among these, E306, E331, H348, and H447, which correspond to the suggested essential catalytic amino acids of the l-fucose isomerase and the l-arabinose isomerase from Escherichia coli, are presumed to be the active-site residues of US100 l-AI. Site-directed mutagenesis confirmed that the mutation of these residues resulted in totally inactive proteins, thus demonstrating their critical role in the enzyme activity. A homology model of US100 l-AI was constructed, and its analysis highlighted another set of residues which may be crucial for the recognition and processing of substrates; hence, these residues were subjected to mutagenesis studies. The replacement of the D308, F329, E351, and H446 amino acids with alanine seriously affected the enzyme activities, and suggestions about the roles of these residues in the catalytic mechanism are given. The mutation F279Q strongly increased the enzyme's affinity for l-fucose and decreased the affinity for l-arabinose compared to that of the wild-type enzyme, showing the implication of this amino acid in substrate recognition.
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Co-expression of l -arabinose isomerase and d -glucose isomerase in E. coli and development of an efficient process producing simultaneously d -tagatose and d -fructose
Enzyme and Microbial Technology, 2007Co-Authors: Moez Rhimi, Ezzedine Ben Messaoud, Mohamed Ali Borgi, Khalifa Ben Khadra, Samir BejarAbstract:To develop a feasible enzymatic process for the concomitant D-tagatose and D-fructose production, the thermostable L-arabinose isomerase of Bacillus stearothermophilus US 100 (L-AI US 100) and the mutant D-glucose isomerase obtained from that of Streptomyces SK (SKGI-A103G) were successfully co-expressed in Escherichia coli HB101 strain. The recombinant cells were immobilized in alginate beads and showed, similarly to the free cells, optimal temperatures for D-galactose and D-glucose isomerisation of 80 and 85 degrees C, respectively. The two Isomerases were optimally active at pH 7.5. Cell entrapment significantly enhanced the acidotolerance of the two Isomerases, as well as their stability at high temperatures. To perform simultaneous isomerisation of D-galactose and D-glucose at 65 degrees C and pH 7.5 in packed-bed bioreactor, cells concentration, dilution rate, productivity and bioconversion rate were optimized to be 32 g/l, 2.6 h(-1), 3 g/l h and 30%, respectively.
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Involvement of alanine 103 residue in kinetic and physicochemical properties of glucose Isomerases from Streptomyces species
Biotechnology Journal, 2007Co-Authors: Mohamed Ali Borgi, Moez Rhimi, Samir BejarAbstract:The Ala103 to Gly mutation, introduced within the glucose isomerase from Streptomyces sp. SK (SKGI) decreased its catalytic efficiency (k(cat)/K(m)) toward D-glucose from 7.1 to 3 mM(-1) min(-1). The reverse counterpart replacement Gly103Ala introduced into the glucose isomerase of Streptomyces olivochromogenes (SOGI) considerably improved its catalytic efficiency to be 6.7 instead of 3.2 mM(-1) min(-1). This later mutation also increased the half-life time of the enzyme from 70 to 95 min at 80 degrees C and mainly modified its pH profile. These results provide evidence that the residue Ala103 plays an essential role in the kinetic and physicochemical properties of glucose Isomerases from Streptomyces species.
Moez Rhimi - One of the best experts on this subject based on the ideXlab platform.
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Bacterial L-arabinose Isomerases: industrial application for D-tagatose production
Recent Patents on DNA & Gene Sequences, 2011Co-Authors: Samira Boudebbouze, Emmanuelle Maguin, Moez RhimiAbstract:D-tagatose is a natural monosaccharide with a low caloric value and has an anti-hyperglycemiant effect. This hexose has potential applications both in pharmaceutical and agro-food industries. However, the use of D-tagatose remains limited by its production cost. Many production procedures including chemical and biological processes were developed and patented. The most profitable production way is based on the use of L-arabinose isomerase which allows the manufacture of D-tagatose with an attractive rate. Future developments are focused on the generation of L-arabinose Isomerases having biochemical properties satisfying the industrial applications. This report provides a brief review of the most recent patents that have been published relating to this area.
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rational design of bacillus stearothermophilus us100 l arabinose isomerase potential applications for d tagatose production
Biochimie, 2009Co-Authors: Moez Rhimi, Richard Haser, Nushin Aghajari, Emmanuelle Maguin, Hichem Chouayekh, Samir BejarAbstract:Abstract l -Arabinose Isomerases catalyze the bioconversion of d -galactose into d -tagatose. With the aim of producing an enzyme optimized for d -tagatose production, three Bacillus stearothermophilus US100 l -arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 °C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0–7.0 and an optimal activity around 50–65 °C, temperatures at which the enzyme was stable without addition of metal ions.
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Rational design of Bacillus stearothermophilus US100 L-arabinose isomerase: potential applications for D-tagatose production
Biochimie, 2009Co-Authors: Moez Rhimi, Richard Haser, Nushin Aghajari, Emmanuelle Maguin, Hichem Chouayekh, Michel Juy, Samir BejarAbstract:L-arabinose Isomerases catalyze the bioconversion of D-galactose into D-tagatose. With the aim of producing an enzyme optimized for D-tagatose production, three Bacillus stearothermophilus US100 L-arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 degrees C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0-7.0 and an optimal activity around 50-65 degrees C, temperatures at which the enzyme was stable without addition of metal ions.
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Bacterial sucrose Isomerases: properties and structural studies
Biologia, 2008Co-Authors: Moez Rhimi, Richard Haser, Nushin AghajariAbstract:Due to their significant role in food industry, sucrose Isomerases are good candidates for rational protein engineering. Hence, specific modifications in order to modify substrate affinity and selectivity, product specificity but also to adapt their catalytic properties to particular industrial process conditions, is interesting. Our work on the structural studies of the sucrose isomerase, MutB, which presents the first structural data available on a trehalulose synthase and the first experimental data on complexed forms of sucrose Isomerases represents a significant advance in the understanding of these enzymes. In this review we give an overview of what is known on biochemical properties and structural aspects of different sucrose Isomerases in particular those reported from bacteria.
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probing the essential catalytic residues and substrate affinity in the thermoactive bacillus stearothermophilus us100 l arabinose isomerase by site directed mutagenesis
Journal of Bacteriology, 2007Co-Authors: Moez Rhimi, Richard Haser, Nushin Aghajari, Samir BejarAbstract:The l-arabinose isomerase (l-AI) from Bacillus stearothermophilus US100 is characterized by its high thermoactivity and catalytic efficiency. Furthermore, as opposed to the majority of l-arabinose Isomerases, this enzyme requires metallic ions for its thermostability rather than for its activity. These features make US100 l-AI attractive as a template for industrial use. Based on previously solved crystal structures and sequence alignments, we identified amino acids that are putatively important for the US100 l-AI isomerization reaction. Among these, E306, E331, H348, and H447, which correspond to the suggested essential catalytic amino acids of the l-fucose isomerase and the l-arabinose isomerase from Escherichia coli, are presumed to be the active-site residues of US100 l-AI. Site-directed mutagenesis confirmed that the mutation of these residues resulted in totally inactive proteins, thus demonstrating their critical role in the enzyme activity. A homology model of US100 l-AI was constructed, and its analysis highlighted another set of residues which may be crucial for the recognition and processing of substrates; hence, these residues were subjected to mutagenesis studies. The replacement of the D308, F329, E351, and H446 amino acids with alanine seriously affected the enzyme activities, and suggestions about the roles of these residues in the catalytic mechanism are given. The mutation F279Q strongly increased the enzyme's affinity for l-fucose and decreased the affinity for l-arabinose compared to that of the wild-type enzyme, showing the implication of this amino acid in substrate recognition.
Richard Haser - One of the best experts on this subject based on the ideXlab platform.
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structural determinants of product specificity of sucrose Isomerases
FEBS Letters, 2009Co-Authors: Stephanie Ravaud, Richard Haser, Xavier Robert, Hildegard Watzlawick, Ralf Mattes, Nushin AghajariAbstract:The healthy sweetener isomaltulose is industrially produced from the conversion of sucrose by the sucrose isomerase SmuA from Protaminobacter rubrum. Crystal structures of SmuA in native and deoxynojirimycin complexed forms completed with modeling studies unravel the characteristics of the isomaltulose synthases catalytic pocket and their substrate binding mode. Comparison with the trehalulose synthase MutB highlights the role of Arg298 and Arg306 active site residues and surface charges in controlling product specificity of sucrose Isomerases (isomaltulose versus trehalulose). The results provide a rationale for the specific design of optimized enzymes.
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rational design of bacillus stearothermophilus us100 l arabinose isomerase potential applications for d tagatose production
Biochimie, 2009Co-Authors: Moez Rhimi, Richard Haser, Nushin Aghajari, Emmanuelle Maguin, Hichem Chouayekh, Samir BejarAbstract:Abstract l -Arabinose Isomerases catalyze the bioconversion of d -galactose into d -tagatose. With the aim of producing an enzyme optimized for d -tagatose production, three Bacillus stearothermophilus US100 l -arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 °C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0–7.0 and an optimal activity around 50–65 °C, temperatures at which the enzyme was stable without addition of metal ions.
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Rational design of Bacillus stearothermophilus US100 L-arabinose isomerase: potential applications for D-tagatose production
Biochimie, 2009Co-Authors: Moez Rhimi, Richard Haser, Nushin Aghajari, Emmanuelle Maguin, Hichem Chouayekh, Michel Juy, Samir BejarAbstract:L-arabinose Isomerases catalyze the bioconversion of D-galactose into D-tagatose. With the aim of producing an enzyme optimized for D-tagatose production, three Bacillus stearothermophilus US100 L-arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 degrees C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0-7.0 and an optimal activity around 50-65 degrees C, temperatures at which the enzyme was stable without addition of metal ions.
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Bacterial sucrose Isomerases: properties and structural studies
Biologia, 2008Co-Authors: Moez Rhimi, Richard Haser, Nushin AghajariAbstract:Due to their significant role in food industry, sucrose Isomerases are good candidates for rational protein engineering. Hence, specific modifications in order to modify substrate affinity and selectivity, product specificity but also to adapt their catalytic properties to particular industrial process conditions, is interesting. Our work on the structural studies of the sucrose isomerase, MutB, which presents the first structural data available on a trehalulose synthase and the first experimental data on complexed forms of sucrose Isomerases represents a significant advance in the understanding of these enzymes. In this review we give an overview of what is known on biochemical properties and structural aspects of different sucrose Isomerases in particular those reported from bacteria.
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probing the essential catalytic residues and substrate affinity in the thermoactive bacillus stearothermophilus us100 l arabinose isomerase by site directed mutagenesis
Journal of Bacteriology, 2007Co-Authors: Moez Rhimi, Richard Haser, Nushin Aghajari, Samir BejarAbstract:The l-arabinose isomerase (l-AI) from Bacillus stearothermophilus US100 is characterized by its high thermoactivity and catalytic efficiency. Furthermore, as opposed to the majority of l-arabinose Isomerases, this enzyme requires metallic ions for its thermostability rather than for its activity. These features make US100 l-AI attractive as a template for industrial use. Based on previously solved crystal structures and sequence alignments, we identified amino acids that are putatively important for the US100 l-AI isomerization reaction. Among these, E306, E331, H348, and H447, which correspond to the suggested essential catalytic amino acids of the l-fucose isomerase and the l-arabinose isomerase from Escherichia coli, are presumed to be the active-site residues of US100 l-AI. Site-directed mutagenesis confirmed that the mutation of these residues resulted in totally inactive proteins, thus demonstrating their critical role in the enzyme activity. A homology model of US100 l-AI was constructed, and its analysis highlighted another set of residues which may be crucial for the recognition and processing of substrates; hence, these residues were subjected to mutagenesis studies. The replacement of the D308, F329, E351, and H446 amino acids with alanine seriously affected the enzyme activities, and suggestions about the roles of these residues in the catalytic mechanism are given. The mutation F279Q strongly increased the enzyme's affinity for l-fucose and decreased the affinity for l-arabinose compared to that of the wild-type enzyme, showing the implication of this amino acid in substrate recognition.
Nushin Aghajari - One of the best experts on this subject based on the ideXlab platform.
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structural determinants of product specificity of sucrose Isomerases
FEBS Letters, 2009Co-Authors: Stephanie Ravaud, Richard Haser, Xavier Robert, Hildegard Watzlawick, Ralf Mattes, Nushin AghajariAbstract:The healthy sweetener isomaltulose is industrially produced from the conversion of sucrose by the sucrose isomerase SmuA from Protaminobacter rubrum. Crystal structures of SmuA in native and deoxynojirimycin complexed forms completed with modeling studies unravel the characteristics of the isomaltulose synthases catalytic pocket and their substrate binding mode. Comparison with the trehalulose synthase MutB highlights the role of Arg298 and Arg306 active site residues and surface charges in controlling product specificity of sucrose Isomerases (isomaltulose versus trehalulose). The results provide a rationale for the specific design of optimized enzymes.
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rational design of bacillus stearothermophilus us100 l arabinose isomerase potential applications for d tagatose production
Biochimie, 2009Co-Authors: Moez Rhimi, Richard Haser, Nushin Aghajari, Emmanuelle Maguin, Hichem Chouayekh, Samir BejarAbstract:Abstract l -Arabinose Isomerases catalyze the bioconversion of d -galactose into d -tagatose. With the aim of producing an enzyme optimized for d -tagatose production, three Bacillus stearothermophilus US100 l -arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 °C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0–7.0 and an optimal activity around 50–65 °C, temperatures at which the enzyme was stable without addition of metal ions.
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Rational design of Bacillus stearothermophilus US100 L-arabinose isomerase: potential applications for D-tagatose production
Biochimie, 2009Co-Authors: Moez Rhimi, Richard Haser, Nushin Aghajari, Emmanuelle Maguin, Hichem Chouayekh, Michel Juy, Samir BejarAbstract:L-arabinose Isomerases catalyze the bioconversion of D-galactose into D-tagatose. With the aim of producing an enzyme optimized for D-tagatose production, three Bacillus stearothermophilus US100 L-arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 degrees C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0-7.0 and an optimal activity around 50-65 degrees C, temperatures at which the enzyme was stable without addition of metal ions.
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Bacterial sucrose Isomerases: properties and structural studies
Biologia, 2008Co-Authors: Moez Rhimi, Richard Haser, Nushin AghajariAbstract:Due to their significant role in food industry, sucrose Isomerases are good candidates for rational protein engineering. Hence, specific modifications in order to modify substrate affinity and selectivity, product specificity but also to adapt their catalytic properties to particular industrial process conditions, is interesting. Our work on the structural studies of the sucrose isomerase, MutB, which presents the first structural data available on a trehalulose synthase and the first experimental data on complexed forms of sucrose Isomerases represents a significant advance in the understanding of these enzymes. In this review we give an overview of what is known on biochemical properties and structural aspects of different sucrose Isomerases in particular those reported from bacteria.
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probing the essential catalytic residues and substrate affinity in the thermoactive bacillus stearothermophilus us100 l arabinose isomerase by site directed mutagenesis
Journal of Bacteriology, 2007Co-Authors: Moez Rhimi, Richard Haser, Nushin Aghajari, Samir BejarAbstract:The l-arabinose isomerase (l-AI) from Bacillus stearothermophilus US100 is characterized by its high thermoactivity and catalytic efficiency. Furthermore, as opposed to the majority of l-arabinose Isomerases, this enzyme requires metallic ions for its thermostability rather than for its activity. These features make US100 l-AI attractive as a template for industrial use. Based on previously solved crystal structures and sequence alignments, we identified amino acids that are putatively important for the US100 l-AI isomerization reaction. Among these, E306, E331, H348, and H447, which correspond to the suggested essential catalytic amino acids of the l-fucose isomerase and the l-arabinose isomerase from Escherichia coli, are presumed to be the active-site residues of US100 l-AI. Site-directed mutagenesis confirmed that the mutation of these residues resulted in totally inactive proteins, thus demonstrating their critical role in the enzyme activity. A homology model of US100 l-AI was constructed, and its analysis highlighted another set of residues which may be crucial for the recognition and processing of substrates; hence, these residues were subjected to mutagenesis studies. The replacement of the D308, F329, E351, and H446 amino acids with alanine seriously affected the enzyme activities, and suggestions about the roles of these residues in the catalytic mechanism are given. The mutation F279Q strongly increased the enzyme's affinity for l-fucose and decreased the affinity for l-arabinose compared to that of the wild-type enzyme, showing the implication of this amino acid in substrate recognition.
S. Bejar - One of the best experts on this subject based on the ideXlab platform.
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Probing the essential catalytic residues and substrate affinity in the thermoactive Bacillus stearothermophilus US100 L-arabinose isomerase by site-directed mutagenesis.
Journal of Bacteriology, 2007Co-Authors: M. Rhimi, M. Juy, N. Aghajari, R. Haser, S. BejarAbstract:The L-arabinose isomerase (L-AI) from Bacillus stearothermophilus US100 is characterized by its high thermoactivity and catalytic efficiency. Furthermore, as opposed to the majority of l-arabinose Isomerases, this enzyme requires metallic ions for its thermostability rather than for its activity. These features make US100 L-AI attractive as a template for industrial use. Based on previously solved crystal structures and sequence alignments, we identified amino acids that are putatively important for the US100 L-AI isomerization reaction. Among these, E306, E331, H348, and H447, which correspond to the suggested essential catalytic amino acids of the L-fucose isomerase and the L-arabinose isomerase from Escherichia coli, are presumed to be the active-site residues of US100 L-AI. Site-directed mutagenesis confirmed that the mutation of these residues resulted in totally inactive proteins, thus demonstrating their critical role in the enzyme activity. A homology model of US100 L-AI was constructed, and its analysis highlighted another set of residues which may be crucial for the recognition and processing of substrates; hence, these residues were subjected to mutagenesis studies. The replacement of the D308, F329, E351, and H446 amino acids with alanine seriously affected the enzyme activities, and suggestions about the roles of these residues in the catalytic mechanism are given. The mutation F279Q strongly increased the enzyme's affinity for L-fucose and decreased the affinity for L-arabinose compared to that of the wild-type enzyme, showing the implication of this amino acid in substrate recognition.The L-arabinose isomerase (L-AI) from Bacillus stearothermophilus US100 is characterized by its high thermoactivity and catalytic efficiency. Furthermore, as opposed to the majority of l-arabinose Isomerases, this enzyme requires metallic ions for its thermostability rather than for its activity. These features make US100 L-AI attractive as a template for industrial use. Based on previously solved crystal structures and sequence alignments, we identified amino acids that are putatively important for the US100 L-AI isomerization reaction. Among these, E306, E331, H348, and H447, which correspond to the suggested essential catalytic amino acids of the L-fucose isomerase and the L-arabinose isomerase from Escherichia coli, are presumed to be the active-site residues of US100 L-AI. Site-directed mutagenesis confirmed that the mutation of these residues resulted in totally inactive proteins, thus demonstrating their critical role in the enzyme activity. A homology model of US100 L-AI was constructed, and its analysis highlighted another set of residues which may be crucial for the recognition and processing of substrates; hence, these residues were subjected to mutagenesis studies. The replacement of the D308, F329, E351, and H446 amino acids with alanine seriously affected the enzyme activities, and suggestions about the roles of these residues in the catalytic mechanism are given. The mutation F279Q strongly increased the enzyme's affinity for L-fucose and decreased the affinity for L-arabinose compared to that of the wild-type enzyme, showing the implication of this amino acid in substrate recognition.
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Glucose isomerase of the Streptomyces sp. SK strain: purification, sequence analysis and implication of alanine 103 residue in the enzyme thermostability and acidotolerance.
Biochimie, 2004Co-Authors: Ma Borgi, R. Haser, K. Srih-belguith, M. Benali, M. Mezghani, S. Tranier, S. BejarAbstract:The glucose isomerase gene (xylA) from the Streptomyces sp. SK strain encodes a 386-amino-acid protein (42.7 kDa) showing extensive identities with many other bacterial glucose Isomerases. We have shown by gel filtration chromatography and SDS-PAGE analysis that the purified recombinant glucose isomerase (SKGI) is a 180 kDa tetramer of four 43 kDa subunits. Sequence inspection revealed that this protein, present some special characteristics like the abundance of hydrophobic residues and some original amino-acid substitutions, which distinguish SKGI from the other GIs previously reported. The presence of an Ala residue at position 103 in SKGI is especially remarkable, since the same amino-acid was found at the equivalent position in the extremely thermostable GIs from Thermus thermophilus and Thermotoga neapolitana; whereas a Gly was found in the majority of less thermostable GIs from Streptomyces. The Ala103Gly mutation, introduced in SKGI, significantly decreases the half-life time at 90 degrees C from 80 to 50 min and also shifts the optimum pH from 6.5 to 7.5. This confirms the implication of the Ala103 residue on SKGI thermostability and activity at low pH. A homology model of SKGI based on the SOGI (that of Streptomyces olivochromogenes) crystal structure has been constructed in order to understand the mutational effects on a molecular scale. Hence, the Ala103Gly mutation, affecting enzyme properties, is presumed to increase molecular flexibility and to destabilize, in particular at elevated temperature, the 91-109 loop that includes the important catalytic residue, Phe94.The glucose isomerase gene (xylA) from the Streptomyces sp. SK strain encodes a 386-amino-acid protein (42.7 kDa) showing extensive identities with many other bacterial glucose Isomerases. We have shown by gel filtration chromatography and SDS-PAGE analysis that the purified recombinant glucose isomerase (SKGI) is a 180 kDa tetramer of four 43 kDa subunits. Sequence inspection revealed that this protein, present some special characteristics like the abundance of hydrophobic residues and some original amino-acid substitutions, which distinguish SKGI from the other GIs previously reported. The presence of an Ala residue at position 103 in SKGI is especially remarkable, since the same amino-acid was found at the equivalent position in the extremely thermostable GIs from Thermus thermophilus and Thermotoga neapolitana; whereas a Gly was found in the majority of less thermostable GIs from Streptomyces. The Ala103Gly mutation, introduced in SKGI, significantly decreases the half-life time at 90 degrees C from 80 to 50 min and also shifts the optimum pH from 6.5 to 7.5. This confirms the implication of the Ala103 residue on SKGI thermostability and activity at low pH. A homology model of SKGI based on the SOGI (that of Streptomyces olivochromogenes) crystal structure has been constructed in order to understand the mutational effects on a molecular scale. Hence, the Ala103Gly mutation, affecting enzyme properties, is presumed to increase molecular flexibility and to destabilize, in particular at elevated temperature, the 91-109 loop that includes the important catalytic residue, Phe94.
