The Experts below are selected from a list of 1527 Experts worldwide ranked by ideXlab platform
Yoshinobu Terada - One of the best experts on this subject based on the ideXlab platform.
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function of second glucan binding site including tyrosines 54 and 101 in Thermus aquaticus amylomaltase
Journal of Bioscience and Bioengineering, 2007Co-Authors: Kazutoshi Fujii, Takashi Kuriki, Yoshinobu Terada, Takeshi Takaha, Hirotaka Minagawa, Jiro Shimada, Hiroki KanekoAbstract:Amylomaltase from Thermus aquaticus catalyzes three types of transglycosylation reaction, as well as a weak hydrolytic reaction of α-1,4 glucan. From our previous study [Fujii et al., Appl. Environ. Microbiol., 71, 5823–5827 (2005)], tyrosine 54 (Y54) was identified as an amino acid controlling the reaction specificity of this enzyme. Since Y54 is not located around the active site but in the proposed second glucan binding site that is 14 A away from catalytic residues, the functions of Y54 and the second glucan binding site are of great interest. In this study, we introduced mutations into another tyrosine (Y101) in the second glucan binding site. The obtained mutated enzymes were subjected to all four types of enzyme assay and the effects of mutations on the reaction specificities of these enzymes were comprehensively investigated. These studies indicated that the amino acid substitution at Y54 or Y101 for removing their aromatic side chain increases cyclization activity (intra-molecular transglycosylation reaction) but decreases disproportionation, coupling and hydrolytic activities (inter-molecular reactions). The superimposition of the reported structures of the enzyme with and without substrate analog revealed the occurrence of a conformational change in which a donor binding site becomes open. From lines of evidence, we conclude that the binding of glucan substrate to the second glucan binding site through an interaction with the aromatic side chains of Y54 and Y101 is a trigger for the enzyme to take a completely active conformation for all four types of activity, but prevents the cyclization reaction to occur since the flexibility of the glucan is restricted by such binding.
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Improvement of Amylomaltase from Thermus aquaticus by Random and Saturation Mutageneses
Journal of Applied Glycoscience, 2005Co-Authors: Kazutoshi Fujii, Takashi Kuriki, Yoshinobu Terada, Takeshi Takaha, Hirotaka Minagawa, Jiro Shimada, Hiroki KanekoAbstract:Amylomaltase (EC 2.4.1.25) from Thermus aquaticus catalyzes an intramolecular transglycosylation of α-1,4 glucan and produces cycloamylose, which is a cyclic α-1,4 glucan with a degree of polymerization of 22 and higher. The amylomaltase has weak but significant hydrolytic activity together with its major transglycosylation activity, which consequently decreases the yield of cycloamylose. To diminish the hydrolytic activity of this enzyme, random mutations are introduced into the gene coding for this enzyme. In the random mutagensesis experiment, it is suggested that tyrosine 54 (Y54), far away from the catalytic site, was involved in hydrolytic activity. In order to investigate the function of Y54, we have performed saturating mutagenesis at Y54 within the amylomaltase and examined the properties of the mutated enzymes. The reaction specificities of the mutated enzymes were surprisingly changed by only one amino acid replacement at Y54. Y54G mutated enzyme had higher cyclization activity in addition to the lower hydrolytic activity. These mutated enzymes also provided useful information to gain further understanding for the activity and the specificity of this enzyme.
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Crystal structure of amylomaltase from Thermus aquaticus, a glycosyltransferase catalysing the production of large cyclic glucans.
Journal of molecular biology, 2000Co-Authors: Ingo Przylas, Koji Tomoo, Yoshinobu Terada, Takeshi Takaha, Kazutoshi Fujii, Wolfram Saenger, Norbert SträterAbstract:Abstract Amylomaltase is involved in the metabolism of starch, one of the most important polysaccharides in nature. A unique feature of amylomaltase is its ability to catalyze the formation of cyclic amylose. In contrast to the well studied cyclodextrin glucanotransferases (CGTases), which synthesize cycloamylose with a ring size (degree of polymerization or DP) of 6-8, the amylomaltase from Thermus aquaticus produces cycloamyloses with a DP of 22 and higher. The crystal structure of amylomaltase from Thermus aquaticus was determined to 2.0 A resolution. It is a member of the α-amylase superfamily of enzymes, whose core structure consists of a (β, α) 8 barrel. In amylomaltase, the 8-fold symmetry of this barrel is disrupted by several insertions between the barrel strands. The largest insertions are between the third and fifth barrel strands, where two insertions form subdomain B1, as well as between the second and third barrel strands, forming the α-helical subdomain B2. Whereas part of subdomain B1 is also present in other enzyme structures of the α-amylase superfamily, subdomain B2 is unique to amylomaltase. Remarkably, the C-terminal domain C, which is present in all related enzymes of the α-amylase family, is missing in amylomaltase. Amylomaltase shows a similar arrangement of the catalytic side-chains (two Asp residues and one Glu residue) as in previously characterized members of the α-amylase superfamily, indicating similar mechanisms of the glycosyl transfer reaction. In amylomaltase, a conserved loop of around eight amino acid residues is partially shielding the active center. This loop, which is well conserved among other amylomaltases, may sterically hinder the formation of small cyclic products.
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Thermus aquaticus atcc 33923 amylomaltase gene cloning and expression and enzyme characterization production of cycloamylose
Applied and Environmental Microbiology, 1999Co-Authors: Yoshinobu Terada, Takeshi Takaha, Kazutoshi Fujii, Shigetaka OkadaAbstract:The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75°C in a 10-min reaction with maltotriose and was stable at temperatures up to 85°C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic α-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coli amylomaltase and the plant D-enzyme.
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Thermus aquaticus atcc 33923 amylomaltase gene cloning and expression and enzyme characterization production of cycloamylose
Applied and Environmental Microbiology, 1999Co-Authors: Yoshinobu Terada, Takeshi Takaha, Kazutoshi Fujii, Shigetaka OkadaAbstract:The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75 degrees C in a 10-min reaction with maltotriose and was stable at temperatures up to 85 degrees C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic alpha-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coli amylomaltase and the plant D-enzyme.
Shigetaka Okada - One of the best experts on this subject based on the ideXlab platform.
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Thermus aquaticus atcc 33923 amylomaltase gene cloning and expression and enzyme characterization production of cycloamylose
Applied and Environmental Microbiology, 1999Co-Authors: Yoshinobu Terada, Takeshi Takaha, Kazutoshi Fujii, Shigetaka OkadaAbstract:The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75°C in a 10-min reaction with maltotriose and was stable at temperatures up to 85°C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic α-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coli amylomaltase and the plant D-enzyme.
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Thermus aquaticus atcc 33923 amylomaltase gene cloning and expression and enzyme characterization production of cycloamylose
Applied and Environmental Microbiology, 1999Co-Authors: Yoshinobu Terada, Takeshi Takaha, Kazutoshi Fujii, Shigetaka OkadaAbstract:The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75 degrees C in a 10-min reaction with maltotriose and was stable at temperatures up to 85 degrees C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic alpha-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coli amylomaltase and the plant D-enzyme.
Vince J. Licata - One of the best experts on this subject based on the ideXlab platform.
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Salt Dependence of DNA binding by Thermus aquaticus and Escherichia coli DNA Polymerases*
2015Co-Authors: Kausiki Datta, Vince J. LicataAbstract:DNA binding properties of the Type 1 DNA poly-merases from Thermus aquaticus (Taq, Klentaq) and Escherichia coli (Klenow) have been examined as a func-tion of [KCl] and [MgCl2]. Full-length Taq and its Klen-taq “large fragment ” behave similarly in all assays. The two different species of polymerases bind DNAwith sub-micromolar affinities in very different salt concentra-tion ranges. Consequently, at similar [KCl] the binding of Klenow is! 3 kcal/mol (150!) tighter than that of Taq/Klentaq to the same DNA. Linkage analysis reveals a net release of 2–3 ions upon DNA binding of Taq/Klen-taq and 4–5 ions upon binding of Klenow. DNA binding of Taq at a higher temperature (60 °C) slightly decreases the ion release. Linkage analysis of binding versus [MgCl2] reports the ultimate release of!1Mg2 " ion upo
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interactions of replication versus repair dna substrates with the pol i dna polymerases from escherichia coli and Thermus aquaticus
Biophysical Chemistry, 2011Co-Authors: Yanling Yang, Vince J. LicataAbstract:Different DNA polymerases partition differently between replication and repair pathways. In this study we examine if two Pol I family polymerases from evolutionarily distant organisms also differ in their preferences for replication versus repair substrates. The DNA binding preferences of Klenow and Klentaq DNA polymerases, from Escherichia coli and Thermus aquaticus respectively, have been studied using a fluorescence competition binding assay. Klenow polymerase binds primed-template DNA (the replication substrate) with up to 50× higher affinity than it binds to nicked DNA, DNA with a 2 base single-stranded gap, blunt-ended DNA, or to a DNA end with a 3' overhang. In contrast, Klentaq binds all of these DNAs almost identically, indicating that Klenow has a stronger ability to discriminate between replication and repair substrates than Klentaq. In contrast, both polymerases bind mismatched primed-template and blunt-ended DNA tighter than they bind matched primed-template DNA, suggesting that these two proteins may share a similar mechanism to identify mismatched DNA, despite the fact that Klentaq has no proofreading ability. In addition, the presence or absence of 5'- or 3'-phosphates has slightly different effects on DNA binding by the two polymerases, but again reinforce Klenow's more effective substrate discrimination capability.
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dna structure selectivity of escherichia coli versus Thermus aquaticus dna polymerase i
Biophysical Journal, 2009Co-Authors: Andy J Wowor, Kausiki Datta, Greg Thompson, Vince J. LicataAbstract:Understanding substrate selection by DNA Polymerase I is important for characterizing the balance between DNA replication and repair for this enzyme in vivo. Due to their sequence and structural similarities, Klenow and Klentaq, the “large fragments” of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, are considered functional homologues. We have examined the DNA binding thermodynamics of Klenow and Klentaq to different DNA structures: single-stranded DNA (ss-DNA), primer-template DNA (pt-DNA), and double-stranded DNA (ds-DNA). The DNA binding affinity trend for Klenow from weakest to tightest binding is ds-DNA < pt-DNA < ss-DNA. This is in contrast to Klentaq's DNA binding trend: ss-DNA < pt-DNA ≈ ds-DNA. Both Klenow and Klentaq released more ions when binding to pt-DNA and ds-DNA than when binding to ss-DNA in KCl buffer. ΔCp is the temperature dependence of the enthalpy of a reaction. Both of these non-sequence specific binding proteins exhibit relatively large heat capacity changes (ΔCp) upon DNA binding. ΔCp values for binding of Klenow and Klentaq to the different DNA structures do not follow the same patterns as the ΔG values for binding, suggesting the balance of electrostatic versus hydrophobic interactions in the binding interfaces also differ between the two species of polymerase. It is also found that Mg2+ significantly shifts the ds-DNA binding affinity of Klenow, but not Klentaq. Mg2+ may be shifting the partitioning between the polymerization and editing sites on Klenow. The differences in DNA structural selectivity of the two polymerases suggest that the in vivo functions of these two supposedly homologous polymerases are different, and that Taq polymerase is more likely to be involved in ds-break repair and end-preservation in vivo. Funded by the NSF and the Louisiana Biomedical Research Network.
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thermodynamics of the binding of Thermus aquaticus dna polymerase to primed template dna
Nucleic Acids Research, 2003Co-Authors: Kausiki Datta, Vince J. LicataAbstract:DNA binding of the Type 1 DNA polymerase from Thermus aquaticus (Taq polymerase) and its Klentaq large fragment domain have been studied as a function of temperature. Equilibrium binding assays were performed from 5 to 70°C using a fluorescence anisotropy assay and from 10 to 60°C using isothermal titration calorimetry. In contrast to the usual behavior of thermophilic proteins at low temperatures, Taq and Klentaq bind DNA with high affinity at temperatures down to 5°C. The affinity is maximal at 40–50°C. The ΔH and ΔS of binding are highly temperature dependent, and the ΔCp of binding is –0.7 to –0.8 kcal/mol K, for both Taq and Klentaq, with good agreement between van’t Hoff and calorimetric values. Such a thermodynamic profile, however, is generally associated with sequence-specific DNA binding and not non- specific binding. Circular dichroism spectra show conformational rearrangements of both the DNA and the protein upon binding. The high ΔCp of Taq/Klentaq DNA binding may be correlated with structure-specific binding in analogy to sequence- specific binding, or may be a general characteristic of proteins that primarily bind non-specifically to DNA. The low temperature DNA binding of Taq/Klentaq is suggested to be a general characteristic of thermophilic DNA binding proteins.
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thermodynamics of the binding of Thermus aquaticus dna polymerase to primed template dna
Nucleic Acids Research, 2003Co-Authors: Kausiki Datta, Vince J. LicataAbstract:DNA binding of the Type 1 DNA polymerase from Thermus aquaticus (Taq polymerase) and its Klentaq large fragment domain have been studied as a function of temperature. Equilibrium binding assays were performed from 5 to 70 degrees C using a fluorescence anisotropy assay and from 10 to 60 degrees C using isothermal titration calorimetry. In contrast to the usual behavior of thermophilic proteins at low temperatures, Taq and Klentaq bind DNA with high affinity at temperatures down to 5 degrees C. The affinity is maximal at 40-50 degrees C. The DeltaH and DeltaS of binding are highly temperature dependent, and the DeltaCp of binding is -0.7 to -0.8 kcal/mol K, for both Taq and Klentaq, with good agreement between van't Hoff and calorimetric values. Such a thermodynamic profile, however, is generally associated with sequence-specific DNA binding and not non- specific binding. Circular dichroism spectra show conformational rearrangements of both the DNA and the protein upon binding. The high DeltaCp of Taq/Klentaq DNA binding may be correlated with structure-specific binding in analogy to sequence- specific binding, or may be a general characteristic of proteins that primarily bind non-specifically to DNA. The low temperature DNA binding of Taq/Klentaq is suggested to be a general characteristic of thermophilic DNA binding proteins.
Kazutoshi Fujii - One of the best experts on this subject based on the ideXlab platform.
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function of second glucan binding site including tyrosines 54 and 101 in Thermus aquaticus amylomaltase
Journal of Bioscience and Bioengineering, 2007Co-Authors: Kazutoshi Fujii, Takashi Kuriki, Yoshinobu Terada, Takeshi Takaha, Hirotaka Minagawa, Jiro Shimada, Hiroki KanekoAbstract:Amylomaltase from Thermus aquaticus catalyzes three types of transglycosylation reaction, as well as a weak hydrolytic reaction of α-1,4 glucan. From our previous study [Fujii et al., Appl. Environ. Microbiol., 71, 5823–5827 (2005)], tyrosine 54 (Y54) was identified as an amino acid controlling the reaction specificity of this enzyme. Since Y54 is not located around the active site but in the proposed second glucan binding site that is 14 A away from catalytic residues, the functions of Y54 and the second glucan binding site are of great interest. In this study, we introduced mutations into another tyrosine (Y101) in the second glucan binding site. The obtained mutated enzymes were subjected to all four types of enzyme assay and the effects of mutations on the reaction specificities of these enzymes were comprehensively investigated. These studies indicated that the amino acid substitution at Y54 or Y101 for removing their aromatic side chain increases cyclization activity (intra-molecular transglycosylation reaction) but decreases disproportionation, coupling and hydrolytic activities (inter-molecular reactions). The superimposition of the reported structures of the enzyme with and without substrate analog revealed the occurrence of a conformational change in which a donor binding site becomes open. From lines of evidence, we conclude that the binding of glucan substrate to the second glucan binding site through an interaction with the aromatic side chains of Y54 and Y101 is a trigger for the enzyme to take a completely active conformation for all four types of activity, but prevents the cyclization reaction to occur since the flexibility of the glucan is restricted by such binding.
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Improvement of Amylomaltase from Thermus aquaticus by Random and Saturation Mutageneses
Journal of Applied Glycoscience, 2005Co-Authors: Kazutoshi Fujii, Takashi Kuriki, Yoshinobu Terada, Takeshi Takaha, Hirotaka Minagawa, Jiro Shimada, Hiroki KanekoAbstract:Amylomaltase (EC 2.4.1.25) from Thermus aquaticus catalyzes an intramolecular transglycosylation of α-1,4 glucan and produces cycloamylose, which is a cyclic α-1,4 glucan with a degree of polymerization of 22 and higher. The amylomaltase has weak but significant hydrolytic activity together with its major transglycosylation activity, which consequently decreases the yield of cycloamylose. To diminish the hydrolytic activity of this enzyme, random mutations are introduced into the gene coding for this enzyme. In the random mutagensesis experiment, it is suggested that tyrosine 54 (Y54), far away from the catalytic site, was involved in hydrolytic activity. In order to investigate the function of Y54, we have performed saturating mutagenesis at Y54 within the amylomaltase and examined the properties of the mutated enzymes. The reaction specificities of the mutated enzymes were surprisingly changed by only one amino acid replacement at Y54. Y54G mutated enzyme had higher cyclization activity in addition to the lower hydrolytic activity. These mutated enzymes also provided useful information to gain further understanding for the activity and the specificity of this enzyme.
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Crystal structure of amylomaltase from Thermus aquaticus, a glycosyltransferase catalysing the production of large cyclic glucans.
Journal of molecular biology, 2000Co-Authors: Ingo Przylas, Koji Tomoo, Yoshinobu Terada, Takeshi Takaha, Kazutoshi Fujii, Wolfram Saenger, Norbert SträterAbstract:Abstract Amylomaltase is involved in the metabolism of starch, one of the most important polysaccharides in nature. A unique feature of amylomaltase is its ability to catalyze the formation of cyclic amylose. In contrast to the well studied cyclodextrin glucanotransferases (CGTases), which synthesize cycloamylose with a ring size (degree of polymerization or DP) of 6-8, the amylomaltase from Thermus aquaticus produces cycloamyloses with a DP of 22 and higher. The crystal structure of amylomaltase from Thermus aquaticus was determined to 2.0 A resolution. It is a member of the α-amylase superfamily of enzymes, whose core structure consists of a (β, α) 8 barrel. In amylomaltase, the 8-fold symmetry of this barrel is disrupted by several insertions between the barrel strands. The largest insertions are between the third and fifth barrel strands, where two insertions form subdomain B1, as well as between the second and third barrel strands, forming the α-helical subdomain B2. Whereas part of subdomain B1 is also present in other enzyme structures of the α-amylase superfamily, subdomain B2 is unique to amylomaltase. Remarkably, the C-terminal domain C, which is present in all related enzymes of the α-amylase family, is missing in amylomaltase. Amylomaltase shows a similar arrangement of the catalytic side-chains (two Asp residues and one Glu residue) as in previously characterized members of the α-amylase superfamily, indicating similar mechanisms of the glycosyl transfer reaction. In amylomaltase, a conserved loop of around eight amino acid residues is partially shielding the active center. This loop, which is well conserved among other amylomaltases, may sterically hinder the formation of small cyclic products.
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Thermus aquaticus atcc 33923 amylomaltase gene cloning and expression and enzyme characterization production of cycloamylose
Applied and Environmental Microbiology, 1999Co-Authors: Yoshinobu Terada, Takeshi Takaha, Kazutoshi Fujii, Shigetaka OkadaAbstract:The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75°C in a 10-min reaction with maltotriose and was stable at temperatures up to 85°C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic α-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coli amylomaltase and the plant D-enzyme.
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Thermus aquaticus atcc 33923 amylomaltase gene cloning and expression and enzyme characterization production of cycloamylose
Applied and Environmental Microbiology, 1999Co-Authors: Yoshinobu Terada, Takeshi Takaha, Kazutoshi Fujii, Shigetaka OkadaAbstract:The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75 degrees C in a 10-min reaction with maltotriose and was stable at temperatures up to 85 degrees C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic alpha-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coli amylomaltase and the plant D-enzyme.
Takeshi Takaha - One of the best experts on this subject based on the ideXlab platform.
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function of second glucan binding site including tyrosines 54 and 101 in Thermus aquaticus amylomaltase
Journal of Bioscience and Bioengineering, 2007Co-Authors: Kazutoshi Fujii, Takashi Kuriki, Yoshinobu Terada, Takeshi Takaha, Hirotaka Minagawa, Jiro Shimada, Hiroki KanekoAbstract:Amylomaltase from Thermus aquaticus catalyzes three types of transglycosylation reaction, as well as a weak hydrolytic reaction of α-1,4 glucan. From our previous study [Fujii et al., Appl. Environ. Microbiol., 71, 5823–5827 (2005)], tyrosine 54 (Y54) was identified as an amino acid controlling the reaction specificity of this enzyme. Since Y54 is not located around the active site but in the proposed second glucan binding site that is 14 A away from catalytic residues, the functions of Y54 and the second glucan binding site are of great interest. In this study, we introduced mutations into another tyrosine (Y101) in the second glucan binding site. The obtained mutated enzymes were subjected to all four types of enzyme assay and the effects of mutations on the reaction specificities of these enzymes were comprehensively investigated. These studies indicated that the amino acid substitution at Y54 or Y101 for removing their aromatic side chain increases cyclization activity (intra-molecular transglycosylation reaction) but decreases disproportionation, coupling and hydrolytic activities (inter-molecular reactions). The superimposition of the reported structures of the enzyme with and without substrate analog revealed the occurrence of a conformational change in which a donor binding site becomes open. From lines of evidence, we conclude that the binding of glucan substrate to the second glucan binding site through an interaction with the aromatic side chains of Y54 and Y101 is a trigger for the enzyme to take a completely active conformation for all four types of activity, but prevents the cyclization reaction to occur since the flexibility of the glucan is restricted by such binding.
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Improvement of Amylomaltase from Thermus aquaticus by Random and Saturation Mutageneses
Journal of Applied Glycoscience, 2005Co-Authors: Kazutoshi Fujii, Takashi Kuriki, Yoshinobu Terada, Takeshi Takaha, Hirotaka Minagawa, Jiro Shimada, Hiroki KanekoAbstract:Amylomaltase (EC 2.4.1.25) from Thermus aquaticus catalyzes an intramolecular transglycosylation of α-1,4 glucan and produces cycloamylose, which is a cyclic α-1,4 glucan with a degree of polymerization of 22 and higher. The amylomaltase has weak but significant hydrolytic activity together with its major transglycosylation activity, which consequently decreases the yield of cycloamylose. To diminish the hydrolytic activity of this enzyme, random mutations are introduced into the gene coding for this enzyme. In the random mutagensesis experiment, it is suggested that tyrosine 54 (Y54), far away from the catalytic site, was involved in hydrolytic activity. In order to investigate the function of Y54, we have performed saturating mutagenesis at Y54 within the amylomaltase and examined the properties of the mutated enzymes. The reaction specificities of the mutated enzymes were surprisingly changed by only one amino acid replacement at Y54. Y54G mutated enzyme had higher cyclization activity in addition to the lower hydrolytic activity. These mutated enzymes also provided useful information to gain further understanding for the activity and the specificity of this enzyme.
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Crystal structure of amylomaltase from Thermus aquaticus, a glycosyltransferase catalysing the production of large cyclic glucans.
Journal of molecular biology, 2000Co-Authors: Ingo Przylas, Koji Tomoo, Yoshinobu Terada, Takeshi Takaha, Kazutoshi Fujii, Wolfram Saenger, Norbert SträterAbstract:Abstract Amylomaltase is involved in the metabolism of starch, one of the most important polysaccharides in nature. A unique feature of amylomaltase is its ability to catalyze the formation of cyclic amylose. In contrast to the well studied cyclodextrin glucanotransferases (CGTases), which synthesize cycloamylose with a ring size (degree of polymerization or DP) of 6-8, the amylomaltase from Thermus aquaticus produces cycloamyloses with a DP of 22 and higher. The crystal structure of amylomaltase from Thermus aquaticus was determined to 2.0 A resolution. It is a member of the α-amylase superfamily of enzymes, whose core structure consists of a (β, α) 8 barrel. In amylomaltase, the 8-fold symmetry of this barrel is disrupted by several insertions between the barrel strands. The largest insertions are between the third and fifth barrel strands, where two insertions form subdomain B1, as well as between the second and third barrel strands, forming the α-helical subdomain B2. Whereas part of subdomain B1 is also present in other enzyme structures of the α-amylase superfamily, subdomain B2 is unique to amylomaltase. Remarkably, the C-terminal domain C, which is present in all related enzymes of the α-amylase family, is missing in amylomaltase. Amylomaltase shows a similar arrangement of the catalytic side-chains (two Asp residues and one Glu residue) as in previously characterized members of the α-amylase superfamily, indicating similar mechanisms of the glycosyl transfer reaction. In amylomaltase, a conserved loop of around eight amino acid residues is partially shielding the active center. This loop, which is well conserved among other amylomaltases, may sterically hinder the formation of small cyclic products.
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Thermus aquaticus atcc 33923 amylomaltase gene cloning and expression and enzyme characterization production of cycloamylose
Applied and Environmental Microbiology, 1999Co-Authors: Yoshinobu Terada, Takeshi Takaha, Kazutoshi Fujii, Shigetaka OkadaAbstract:The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75°C in a 10-min reaction with maltotriose and was stable at temperatures up to 85°C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic α-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coli amylomaltase and the plant D-enzyme.
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Thermus aquaticus atcc 33923 amylomaltase gene cloning and expression and enzyme characterization production of cycloamylose
Applied and Environmental Microbiology, 1999Co-Authors: Yoshinobu Terada, Takeshi Takaha, Kazutoshi Fujii, Shigetaka OkadaAbstract:The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75 degrees C in a 10-min reaction with maltotriose and was stable at temperatures up to 85 degrees C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic alpha-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coli amylomaltase and the plant D-enzyme.