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A Maujean - One of the best experts on this subject based on the ideXlab platform.

  • alpha amylase isoform pattern changes during the winter season in the winter resting stem inter nodes of vitis vinifera
    Plant Physiology and Biochemistry, 1997
    Co-Authors: P Berbezy, Laurent Legendre, A Maujean
    Abstract:

    In order to better characterize the potential role played by Alpha-Amylases (EC 3.2.1.1) during the winter, changes in Alpha-Amylase isoform pattern were first analyzed as the season progressed. Crude protein extracts from winter-resting stems of the grapevine plant Vitis vinifera were thus separated by isoelectric focusing under native conditions. Two groups of Alpha-Amylases isoforms could be seen. One group was characterized by high pI's (around 6.5) and was mainly expressed during the first phase of the winter (post-dormancy period) whereas the second one had lower pI's (around 4.5) and was expressed during the second half of the winter (pre-bursting period). Alpha-Amylase activity increases corresponded to parallel losses in starch content and rises in the concentration of some soluble sugars in the woods studied. A direct assessment of the influence of drops in temperature on Alpha-Amylase activity changes and isoforms expression was obtained after placing grapevine stems at different laboratory-controlled temperatures. Lowering the temperature from 12°C to 4°C led to the maximum induction of both groups of Alpha-Amylase isoforms with a parallel hydrolysis of starch and release of some soluble sugars. Interestingly, a controlled temperature drop from 12°C to 4°C also led to a maximum increase in sucrose content whereas a further drop to sub-freezing temperatures (-15°C) was necessary to trigger raffinose accumulation.

  • alpha amylase isoform pattern changes during the winter season in the winter resting stem inter nodes of vitis vinifera
    Plant Physiology and Biochemistry, 1997
    Co-Authors: P Berbezy, Laurent Legendre, A Maujean
    Abstract:

    In order to better characterize the potential role played by Alpha-Amylases (EC 3.2.1.1) during the winter, changes in Alpha-Amylase isoform pattern were first analyzed as the season progressed. Crude protein extracts from winter-resting stems of the grapevine plant Vitis vinifera were thus separated by isoelectric focusing under native conditions. Two groups of Alpha-Amylases isoforms could be seen. One group was characterized by high pI's (around 6.5) and was mainly expressed during the first phase of the winter (post-dormancy period) whereas the second one had lower pI's (around 4.5) and was expressed during the second half of the winter (pre-bursting period). Alpha-Amylase activity increases corresponded to parallel losses in starch content and rises in the concentration of some soluble sugars in the woods studied. A direct assessment of the influence of drops in temperature on Alpha-Amylase activity changes and isoforms expression was obtained after placing grapevine stems at different laboratory-controlled temperatures. Lowering the temperature from 12°C to 4°C led to the maximum induction of both groups of Alpha-Amylase isoforms with a parallel hydrolysis of starch and release of some soluble sugars. Interestingly, a controlled temperature drop from 12°C to 4°C also led to a maximum increase in sucrose content whereas a further drop to sub-freezing temperatures (-15°C) was necessary to trigger raffinose accumulation.

N. Aghajari - One of the best experts on this subject based on the ideXlab platform.

  • Oligosaccharide binding to barley Alpha-Amylase 1.
    Journal of Biological Chemistry, 2005
    Co-Authors: X. Robert, R. Haser, H. Mori, B. Svensson, N. Aghajari
    Abstract:

    Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley Alpha-Amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley Alpha-Amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely -7 through +2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley Alpha-Amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1D180A) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers."Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley Alpha-Amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley Alpha-Amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely -7 through +2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley Alpha-Amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1D180A) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers.

  • The structure of barley Alpha-Amylase isozyme 1 reveals a novel role of domain C in substrate recognition and binding: a pair of sugar tongs.
    Structure, 2003
    Co-Authors: X. Robert, R. Haser, B. Svensson, Te Gottschalk, F. Ratajczak, H. Driguez, N. Aghajari
    Abstract:

    Though the three-dimensional structures of barley Alpha-Amylase isozymes AMY1 and AMY2 are very similar, they differ remarkably from each other in their affinity for Ca(2+) and when interacting with substrate analogs. A surface site recognizing maltooligosaccharides, not earlier reported for other Alpha-Amylases and probably associated with the different activity of AMY1 and AMY2 toward starch granules, has been identified. It is located in the C-terminal part of the enzyme and, thus, highlights a potential role of domain C. In order to scrutinize the possible biological significance of this domain in Alpha-Amylases, a thorough comparison of their three-dimensional structures was conducted. An additional role for an earlier-identified starch granule binding surface site is proposed, and a new calcium ion is reported.Though the three-dimensional structures of barley Alpha-Amylase isozymes AMY1 and AMY2 are very similar, they differ remarkably from each other in their affinity for Ca(2+) and when interacting with substrate analogs. A surface site recognizing maltooligosaccharides, not earlier reported for other Alpha-Amylases and probably associated with the different activity of AMY1 and AMY2 toward starch granules, has been identified. It is located in the C-terminal part of the enzyme and, thus, highlights a potential role of domain C. In order to scrutinize the possible biological significance of this domain in Alpha-Amylases, a thorough comparison of their three-dimensional structures was conducted. An additional role for an earlier-identified starch granule binding surface site is proposed, and a new calcium ion is reported.

  • Structural basis of Alpha-Amylase activation by chloride.
    Protein Science, 2002
    Co-Authors: N. Aghajari, G. Feller, C. Gerday, R. Haser
    Abstract:

    To further investigate the mechanism and function of allosteric activation by chloride in some Alpha-Amylases, the structure of the bacterial Alpha-Amylase from the psychrophilic micro-organism Pseudoalteromonas haloplanktis in complex with nitrate has been solved at 2.1 A degrees, as well as the structure of the mutants Lys300Gln (2.5 A degrees ) and Lys300Arg (2.25 A degrees ). Nitrate binds strongly to Alpha-Amylase but is a weak activator. Mutation of the critical chloride ligand Lys300 into Gln results in a chloride-independent enzyme, whereas the mutation into Arg mimics the binding site as is found in animal Alpha-Amylases with, however, a lower affinity for chloride. These structures reveal that the triangular conformation of the chloride ligands and the nearly equatorial coordination allow the perfect accommodation of planar trigonal monovalent anions such as NO3-, explaining their unusual strong binding. It is also shown that a localized negative charge such as that of Cl-, rather than a delocalized charge as in the case of nitrate, is essential for maximal activation. The chloride-free mutant Lys300Gln indicates that chloride is not mandatory for the catalytic mechanism but strongly increases the reactivity at the active site. Disappearance of the putative catalytic water molecule in this weakly active mutant supports the view that chloride helps to polarize the hydrolytic water molecule and enhances the rate of the second step in the catalytic reaction.To further investigate the mechanism and function of allosteric activation by chloride in some Alpha-Amylases, the structure of the bacterial Alpha-Amylase from the psychrophilic micro-organism Pseudoalteromonas haloplanktis in complex with nitrate has been solved at 2.1 A degrees, as well as the structure of the mutants Lys300Gln (2.5 A degrees ) and Lys300Arg (2.25 A degrees ). Nitrate binds strongly to Alpha-Amylase but is a weak activator. Mutation of the critical chloride ligand Lys300 into Gln results in a chloride-independent enzyme, whereas the mutation into Arg mimics the binding site as is found in animal Alpha-Amylases with, however, a lower affinity for chloride. These structures reveal that the triangular conformation of the chloride ligands and the nearly equatorial coordination allow the perfect accommodation of planar trigonal monovalent anions such as NO3-, explaining their unusual strong binding. It is also shown that a localized negative charge such as that of Cl-, rather than a delocalized charge as in the case of nitrate, is essential for maximal activation. The chloride-free mutant Lys300Gln indicates that chloride is not mandatory for the catalytic mechanism but strongly increases the reactivity at the active site. Disappearance of the putative catalytic water molecule in this weakly active mutant supports the view that chloride helps to polarize the hydrolytic water molecule and enhances the rate of the second step in the catalytic reaction.

  • crystal structures of the psychrophilic alpha amylase from alteromonas haloplanctis in its native form and complexed with an inhibitor
    Protein Science, 1998
    Co-Authors: N. Aghajari, Georges Feller, Charles Gerday, R. Haser
    Abstract:

    Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the Alpha-Amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known Alpha-Amylases of various origins with a surprisingly greatest similarity to mammalian Alpha-Amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent Alpha-Amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted Alpha-Amylase.

  • Crystal structures of the psychrophilic Alpha-Amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor.
    Protein Science, 1998
    Co-Authors: N. Aghajari, Georges Feller, Charles Gerday, R. Haser
    Abstract:

    Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the Alpha-Amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known Alpha-Amylases of various origins with a surprisingly greatest similarity to mammalian Alpha-Amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent Alpha-Amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted Alpha-Amylase.Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the Alpha-Amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known Alpha-Amylases of various origins with a surprisingly greatest similarity to mammalian Alpha-Amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent Alpha-Amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted Alpha-Amylase.

R. Haser - One of the best experts on this subject based on the ideXlab platform.

  • Oligosaccharide binding to barley Alpha-Amylase 1.
    Journal of Biological Chemistry, 2005
    Co-Authors: X. Robert, R. Haser, H. Mori, B. Svensson, N. Aghajari
    Abstract:

    Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley Alpha-Amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley Alpha-Amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely -7 through +2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley Alpha-Amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1D180A) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers."Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley Alpha-Amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley Alpha-Amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely -7 through +2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley Alpha-Amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1D180A) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers.

  • The structure of barley Alpha-Amylase isozyme 1 reveals a novel role of domain C in substrate recognition and binding: a pair of sugar tongs.
    Structure, 2003
    Co-Authors: X. Robert, R. Haser, B. Svensson, Te Gottschalk, F. Ratajczak, H. Driguez, N. Aghajari
    Abstract:

    Though the three-dimensional structures of barley Alpha-Amylase isozymes AMY1 and AMY2 are very similar, they differ remarkably from each other in their affinity for Ca(2+) and when interacting with substrate analogs. A surface site recognizing maltooligosaccharides, not earlier reported for other Alpha-Amylases and probably associated with the different activity of AMY1 and AMY2 toward starch granules, has been identified. It is located in the C-terminal part of the enzyme and, thus, highlights a potential role of domain C. In order to scrutinize the possible biological significance of this domain in Alpha-Amylases, a thorough comparison of their three-dimensional structures was conducted. An additional role for an earlier-identified starch granule binding surface site is proposed, and a new calcium ion is reported.Though the three-dimensional structures of barley Alpha-Amylase isozymes AMY1 and AMY2 are very similar, they differ remarkably from each other in their affinity for Ca(2+) and when interacting with substrate analogs. A surface site recognizing maltooligosaccharides, not earlier reported for other Alpha-Amylases and probably associated with the different activity of AMY1 and AMY2 toward starch granules, has been identified. It is located in the C-terminal part of the enzyme and, thus, highlights a potential role of domain C. In order to scrutinize the possible biological significance of this domain in Alpha-Amylases, a thorough comparison of their three-dimensional structures was conducted. An additional role for an earlier-identified starch granule binding surface site is proposed, and a new calcium ion is reported.

  • Structural basis of Alpha-Amylase activation by chloride.
    Protein Science, 2002
    Co-Authors: N. Aghajari, G. Feller, C. Gerday, R. Haser
    Abstract:

    To further investigate the mechanism and function of allosteric activation by chloride in some Alpha-Amylases, the structure of the bacterial Alpha-Amylase from the psychrophilic micro-organism Pseudoalteromonas haloplanktis in complex with nitrate has been solved at 2.1 A degrees, as well as the structure of the mutants Lys300Gln (2.5 A degrees ) and Lys300Arg (2.25 A degrees ). Nitrate binds strongly to Alpha-Amylase but is a weak activator. Mutation of the critical chloride ligand Lys300 into Gln results in a chloride-independent enzyme, whereas the mutation into Arg mimics the binding site as is found in animal Alpha-Amylases with, however, a lower affinity for chloride. These structures reveal that the triangular conformation of the chloride ligands and the nearly equatorial coordination allow the perfect accommodation of planar trigonal monovalent anions such as NO3-, explaining their unusual strong binding. It is also shown that a localized negative charge such as that of Cl-, rather than a delocalized charge as in the case of nitrate, is essential for maximal activation. The chloride-free mutant Lys300Gln indicates that chloride is not mandatory for the catalytic mechanism but strongly increases the reactivity at the active site. Disappearance of the putative catalytic water molecule in this weakly active mutant supports the view that chloride helps to polarize the hydrolytic water molecule and enhances the rate of the second step in the catalytic reaction.To further investigate the mechanism and function of allosteric activation by chloride in some Alpha-Amylases, the structure of the bacterial Alpha-Amylase from the psychrophilic micro-organism Pseudoalteromonas haloplanktis in complex with nitrate has been solved at 2.1 A degrees, as well as the structure of the mutants Lys300Gln (2.5 A degrees ) and Lys300Arg (2.25 A degrees ). Nitrate binds strongly to Alpha-Amylase but is a weak activator. Mutation of the critical chloride ligand Lys300 into Gln results in a chloride-independent enzyme, whereas the mutation into Arg mimics the binding site as is found in animal Alpha-Amylases with, however, a lower affinity for chloride. These structures reveal that the triangular conformation of the chloride ligands and the nearly equatorial coordination allow the perfect accommodation of planar trigonal monovalent anions such as NO3-, explaining their unusual strong binding. It is also shown that a localized negative charge such as that of Cl-, rather than a delocalized charge as in the case of nitrate, is essential for maximal activation. The chloride-free mutant Lys300Gln indicates that chloride is not mandatory for the catalytic mechanism but strongly increases the reactivity at the active site. Disappearance of the putative catalytic water molecule in this weakly active mutant supports the view that chloride helps to polarize the hydrolytic water molecule and enhances the rate of the second step in the catalytic reaction.

  • crystal structures of the psychrophilic alpha amylase from alteromonas haloplanctis in its native form and complexed with an inhibitor
    Protein Science, 1998
    Co-Authors: N. Aghajari, Georges Feller, Charles Gerday, R. Haser
    Abstract:

    Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the Alpha-Amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known Alpha-Amylases of various origins with a surprisingly greatest similarity to mammalian Alpha-Amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent Alpha-Amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted Alpha-Amylase.

  • Crystal structures of the psychrophilic Alpha-Amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor.
    Protein Science, 1998
    Co-Authors: N. Aghajari, Georges Feller, Charles Gerday, R. Haser
    Abstract:

    Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the Alpha-Amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known Alpha-Amylases of various origins with a surprisingly greatest similarity to mammalian Alpha-Amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent Alpha-Amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted Alpha-Amylase.Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the Alpha-Amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known Alpha-Amylases of various origins with a surprisingly greatest similarity to mammalian Alpha-Amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent Alpha-Amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted Alpha-Amylase.

Andrea Brandolini - One of the best experts on this subject based on the ideXlab platform.

  • polyphenol oxidase alpha amylase and beta amylase activities of triticum monococcum triticum turgidum and triticum aestivum a two year study
    Journal of Cereal Science, 2013
    Co-Authors: Alyssa Hidalgo, Marta Brusco, L Plizzari, Andrea Brandolini
    Abstract:

    Abstract Enzymatic activity often reduces the nutritional value of wheat flour during food manufacturing, causing compound degradation and/or heat damage. The choice of wheat varieties with low enzymatic activity could therefore help to preserve the nutritional quality of food. The aim of this research was to evaluate polyphenol oxidase, Alpha-Amylase and beta-amylase activities in whole meal flours of 59 accessions belonging to different wheat species and subspecies, cropped in two years. The extraction pH (7.0), reaction pH (5.5) and reaction temperature (45 °C) were determined by preliminary trials. The ANOVA highlighted significant differences for all enzymes among species/subspecies and, for amylases, between cropping years; however, the year influence was overwhelming only for Alpha-Amylase. Einkorn showed the highest polyphenol oxidase (362.1 ± 9.46 U/g DM) as well as the lowest Alpha-Amylase (0.20 ± 0.006 CU/g DM) and beta-amylase (12.0 ± 0.36 B3U/g DM) activities. The embryo/scutellum had the highest polyphenol oxidase and Alpha-Amylase values, followed by the bran and the endosperm; in contrast, beta-amylase was evenly distributed in the bran and the endosperm, and was absent in the embryo/scutellum.

  • Polyphenol oxidase, Alpha-Amylase and beta-amylase activities of Triticum monococcum, Triticum turgidum and Triticum aestivum: A two-year study
    Journal of Cereal Science, 2013
    Co-Authors: Alyssa Hidalgo, Marta Brusco, L Plizzari, Andrea Brandolini
    Abstract:

    Abstract Enzymatic activity often reduces the nutritional value of wheat flour during food manufacturing, causing compound degradation and/or heat damage. The choice of wheat varieties with low enzymatic activity could therefore help to preserve the nutritional quality of food. The aim of this research was to evaluate polyphenol oxidase, Alpha-Amylase and beta-amylase activities in whole meal flours of 59 accessions belonging to different wheat species and subspecies, cropped in two years. The extraction pH (7.0), reaction pH (5.5) and reaction temperature (45 °C) were determined by preliminary trials. The ANOVA highlighted significant differences for all enzymes among species/subspecies and, for amylases, between cropping years; however, the year influence was overwhelming only for Alpha-Amylase. Einkorn showed the highest polyphenol oxidase (362.1 ± 9.46 U/g DM) as well as the lowest Alpha-Amylase (0.20 ± 0.006 CU/g DM) and beta-amylase (12.0 ± 0.36 B3U/g DM) activities. The embryo/scutellum had the highest polyphenol oxidase and Alpha-Amylase values, followed by the bran and the endosperm; in contrast, beta-amylase was evenly distributed in the bran and the endosperm, and was absent in the embryo/scutellum.

P Berbezy - One of the best experts on this subject based on the ideXlab platform.

  • alpha amylase isoform pattern changes during the winter season in the winter resting stem inter nodes of vitis vinifera
    Plant Physiology and Biochemistry, 1997
    Co-Authors: P Berbezy, Laurent Legendre, A Maujean
    Abstract:

    In order to better characterize the potential role played by Alpha-Amylases (EC 3.2.1.1) during the winter, changes in Alpha-Amylase isoform pattern were first analyzed as the season progressed. Crude protein extracts from winter-resting stems of the grapevine plant Vitis vinifera were thus separated by isoelectric focusing under native conditions. Two groups of Alpha-Amylases isoforms could be seen. One group was characterized by high pI's (around 6.5) and was mainly expressed during the first phase of the winter (post-dormancy period) whereas the second one had lower pI's (around 4.5) and was expressed during the second half of the winter (pre-bursting period). Alpha-Amylase activity increases corresponded to parallel losses in starch content and rises in the concentration of some soluble sugars in the woods studied. A direct assessment of the influence of drops in temperature on Alpha-Amylase activity changes and isoforms expression was obtained after placing grapevine stems at different laboratory-controlled temperatures. Lowering the temperature from 12°C to 4°C led to the maximum induction of both groups of Alpha-Amylase isoforms with a parallel hydrolysis of starch and release of some soluble sugars. Interestingly, a controlled temperature drop from 12°C to 4°C also led to a maximum increase in sucrose content whereas a further drop to sub-freezing temperatures (-15°C) was necessary to trigger raffinose accumulation.

  • alpha amylase isoform pattern changes during the winter season in the winter resting stem inter nodes of vitis vinifera
    Plant Physiology and Biochemistry, 1997
    Co-Authors: P Berbezy, Laurent Legendre, A Maujean
    Abstract:

    In order to better characterize the potential role played by Alpha-Amylases (EC 3.2.1.1) during the winter, changes in Alpha-Amylase isoform pattern were first analyzed as the season progressed. Crude protein extracts from winter-resting stems of the grapevine plant Vitis vinifera were thus separated by isoelectric focusing under native conditions. Two groups of Alpha-Amylases isoforms could be seen. One group was characterized by high pI's (around 6.5) and was mainly expressed during the first phase of the winter (post-dormancy period) whereas the second one had lower pI's (around 4.5) and was expressed during the second half of the winter (pre-bursting period). Alpha-Amylase activity increases corresponded to parallel losses in starch content and rises in the concentration of some soluble sugars in the woods studied. A direct assessment of the influence of drops in temperature on Alpha-Amylase activity changes and isoforms expression was obtained after placing grapevine stems at different laboratory-controlled temperatures. Lowering the temperature from 12°C to 4°C led to the maximum induction of both groups of Alpha-Amylase isoforms with a parallel hydrolysis of starch and release of some soluble sugars. Interestingly, a controlled temperature drop from 12°C to 4°C also led to a maximum increase in sucrose content whereas a further drop to sub-freezing temperatures (-15°C) was necessary to trigger raffinose accumulation.