Arabidopsis thaliana AMY3 Is a Unique Redox-regulated Chloroplastic α-Amylase

David Seung, Matthias Thalmann, Francesca Sparla, Maher Abou Hachem, Sang Kyu Lee, Emmanuelle Issakidis-Bourguet, Birte Svensson, Samuel C. Zeeman, Diana Santelia

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. In vascular plants, α-amylases can be classified into three subfamilies. Arabidopsis has one member of each subfamily. Among them, only AtAMY3 is localized in the chloroplast. We expressed and purified AtAMY3 from Escherichia coli and carried out a biochemical characterization of the protein to find factors that regulate its activity. Recombinant AtAMY3 was active toward both insoluble starch granules and soluble substrates, with a strong preference for β-limit dextrin over amylopectin. Activity was shown to be dependent on a conserved aspartic acid residue (Asp666), identified as the catalytic nucleophile in other plant α-amylases such as the barley AMY1. AtAMY3 released small linear and branched glucans from Arabidopsis starch granules, and the proportion of branched glucans increased after the predigestion of starch with a β-amylase. Optimal rates of starch digestion in vitro was achieved when both AtAMY3 and β-amylase activities were present, suggesting that the two enzymes work synergistically at the granule surface. We also found that AtAMY3 has unique properties among other characterized plant α-amylases, with a pH optimum of 7.5–8, appropriate for activity in the chloroplast stroma. AtAMY3 is also redox-regulated, and the inactive oxidized form of AtAMY3 could be reactivated by reduced thioredoxins. Site-directed mutagenesis combined with mass spectrometry analysis showed that a disulfide bridge between Cys499 and Cys587 is central to this regulation. This work provides new insights into how α-amylase activity may be regulated in the chloroplast.
    Original languageEnglish
    JournalJournal of Biological Chemistry
    Volume288
    Issue number47
    Pages (from-to)33620-33633
    ISSN0021-9258
    DOIs
    Publication statusPublished - 2013

    Cite this

    Seung, D., Thalmann, M., Sparla, F., Abou Hachem, M., Lee, S. K., Issakidis-Bourguet, E., ... Santelia, D. (2013). Arabidopsis thaliana AMY3 Is a Unique Redox-regulated Chloroplastic α-Amylase. Journal of Biological Chemistry, 288(47), 33620-33633. https://doi.org/10.1074/jbc.M113.514794
    Seung, David ; Thalmann, Matthias ; Sparla, Francesca ; Abou Hachem, Maher ; Lee, Sang Kyu ; Issakidis-Bourguet, Emmanuelle ; Svensson, Birte ; Zeeman, Samuel C. ; Santelia, Diana. / Arabidopsis thaliana AMY3 Is a Unique Redox-regulated Chloroplastic α-Amylase. In: Journal of Biological Chemistry. 2013 ; Vol. 288, No. 47. pp. 33620-33633.
    @article{34d8b255f5cd4d8ab7348b68583dd110,
    title = "Arabidopsis thaliana AMY3 Is a Unique Redox-regulated Chloroplastic α-Amylase",
    abstract = "α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. In vascular plants, α-amylases can be classified into three subfamilies. Arabidopsis has one member of each subfamily. Among them, only AtAMY3 is localized in the chloroplast. We expressed and purified AtAMY3 from Escherichia coli and carried out a biochemical characterization of the protein to find factors that regulate its activity. Recombinant AtAMY3 was active toward both insoluble starch granules and soluble substrates, with a strong preference for β-limit dextrin over amylopectin. Activity was shown to be dependent on a conserved aspartic acid residue (Asp666), identified as the catalytic nucleophile in other plant α-amylases such as the barley AMY1. AtAMY3 released small linear and branched glucans from Arabidopsis starch granules, and the proportion of branched glucans increased after the predigestion of starch with a β-amylase. Optimal rates of starch digestion in vitro was achieved when both AtAMY3 and β-amylase activities were present, suggesting that the two enzymes work synergistically at the granule surface. We also found that AtAMY3 has unique properties among other characterized plant α-amylases, with a pH optimum of 7.5–8, appropriate for activity in the chloroplast stroma. AtAMY3 is also redox-regulated, and the inactive oxidized form of AtAMY3 could be reactivated by reduced thioredoxins. Site-directed mutagenesis combined with mass spectrometry analysis showed that a disulfide bridge between Cys499 and Cys587 is central to this regulation. This work provides new insights into how α-amylase activity may be regulated in the chloroplast.",
    author = "David Seung and Matthias Thalmann and Francesca Sparla and {Abou Hachem}, Maher and Lee, {Sang Kyu} and Emmanuelle Issakidis-Bourguet and Birte Svensson and Zeeman, {Samuel C.} and Diana Santelia",
    year = "2013",
    doi = "10.1074/jbc.M113.514794",
    language = "English",
    volume = "288",
    pages = "33620--33633",
    journal = "Journal of Biological Chemistry",
    issn = "0021-9258",
    publisher = "American Society for Biochemistry and Molecular Biology, Inc.",
    number = "47",

    }

    Seung, D, Thalmann, M, Sparla, F, Abou Hachem, M, Lee, SK, Issakidis-Bourguet, E, Svensson, B, Zeeman, SC & Santelia, D 2013, 'Arabidopsis thaliana AMY3 Is a Unique Redox-regulated Chloroplastic α-Amylase', Journal of Biological Chemistry, vol. 288, no. 47, pp. 33620-33633. https://doi.org/10.1074/jbc.M113.514794

    Arabidopsis thaliana AMY3 Is a Unique Redox-regulated Chloroplastic α-Amylase. / Seung, David; Thalmann, Matthias; Sparla, Francesca; Abou Hachem, Maher ; Lee, Sang Kyu; Issakidis-Bourguet, Emmanuelle; Svensson, Birte; Zeeman, Samuel C.; Santelia, Diana.

    In: Journal of Biological Chemistry, Vol. 288, No. 47, 2013, p. 33620-33633.

    Research output: Contribution to journalJournal articleResearchpeer-review

    TY - JOUR

    T1 - Arabidopsis thaliana AMY3 Is a Unique Redox-regulated Chloroplastic α-Amylase

    AU - Seung, David

    AU - Thalmann, Matthias

    AU - Sparla, Francesca

    AU - Abou Hachem, Maher

    AU - Lee, Sang Kyu

    AU - Issakidis-Bourguet, Emmanuelle

    AU - Svensson, Birte

    AU - Zeeman, Samuel C.

    AU - Santelia, Diana

    PY - 2013

    Y1 - 2013

    N2 - α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. In vascular plants, α-amylases can be classified into three subfamilies. Arabidopsis has one member of each subfamily. Among them, only AtAMY3 is localized in the chloroplast. We expressed and purified AtAMY3 from Escherichia coli and carried out a biochemical characterization of the protein to find factors that regulate its activity. Recombinant AtAMY3 was active toward both insoluble starch granules and soluble substrates, with a strong preference for β-limit dextrin over amylopectin. Activity was shown to be dependent on a conserved aspartic acid residue (Asp666), identified as the catalytic nucleophile in other plant α-amylases such as the barley AMY1. AtAMY3 released small linear and branched glucans from Arabidopsis starch granules, and the proportion of branched glucans increased after the predigestion of starch with a β-amylase. Optimal rates of starch digestion in vitro was achieved when both AtAMY3 and β-amylase activities were present, suggesting that the two enzymes work synergistically at the granule surface. We also found that AtAMY3 has unique properties among other characterized plant α-amylases, with a pH optimum of 7.5–8, appropriate for activity in the chloroplast stroma. AtAMY3 is also redox-regulated, and the inactive oxidized form of AtAMY3 could be reactivated by reduced thioredoxins. Site-directed mutagenesis combined with mass spectrometry analysis showed that a disulfide bridge between Cys499 and Cys587 is central to this regulation. This work provides new insights into how α-amylase activity may be regulated in the chloroplast.

    AB - α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. In vascular plants, α-amylases can be classified into three subfamilies. Arabidopsis has one member of each subfamily. Among them, only AtAMY3 is localized in the chloroplast. We expressed and purified AtAMY3 from Escherichia coli and carried out a biochemical characterization of the protein to find factors that regulate its activity. Recombinant AtAMY3 was active toward both insoluble starch granules and soluble substrates, with a strong preference for β-limit dextrin over amylopectin. Activity was shown to be dependent on a conserved aspartic acid residue (Asp666), identified as the catalytic nucleophile in other plant α-amylases such as the barley AMY1. AtAMY3 released small linear and branched glucans from Arabidopsis starch granules, and the proportion of branched glucans increased after the predigestion of starch with a β-amylase. Optimal rates of starch digestion in vitro was achieved when both AtAMY3 and β-amylase activities were present, suggesting that the two enzymes work synergistically at the granule surface. We also found that AtAMY3 has unique properties among other characterized plant α-amylases, with a pH optimum of 7.5–8, appropriate for activity in the chloroplast stroma. AtAMY3 is also redox-regulated, and the inactive oxidized form of AtAMY3 could be reactivated by reduced thioredoxins. Site-directed mutagenesis combined with mass spectrometry analysis showed that a disulfide bridge between Cys499 and Cys587 is central to this regulation. This work provides new insights into how α-amylase activity may be regulated in the chloroplast.

    U2 - 10.1074/jbc.M113.514794

    DO - 10.1074/jbc.M113.514794

    M3 - Journal article

    VL - 288

    SP - 33620

    EP - 33633

    JO - Journal of Biological Chemistry

    JF - Journal of Biological Chemistry

    SN - 0021-9258

    IS - 47

    ER -