The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel alpha-glucosides through reverse phosphorolysis by maltose phosphorylase

Hiroyuki Nakai, Martin Baumann, B.O. Petersen, Y. Westphal, H. Schols, Adiphol Dilokpimol, Maher Abou Hachem, S.J. Lahtinen, Jens Øllgaard Duus, Birte Svensson

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI-GalR family. Enzymatic properties are described for recombinant maltose phosphorylase (MalP) of glycoside hydrolase family 65 (GH65), which is encoded by malP (GenBank: AAV43670.1) of this gene cluster and produced in Escherichia coli. MalP catalyses phosphorolysis of maltose with inversion of the anomeric configuration releasing beta-glucose 1-phosphate (beta-Glc 1-P) and glucose. The broad specificity of the aglycone binding site was demonstrated by products formed in reverse phosphorolysis using various carbohydrate acceptor substrates and beta-Glc 1-P as the donor. MalP showed strong preference for monosaccharide acceptors with equatorial 3-OH and 4-OH, such as glucose and mannose, and also reacted with 2-deoxy glucosamine and 2-deoxy N-acetyl glucosamine. By contrast, none of the tested di- and trisaccharides served as acceptors. Disaccharide yields obtained from 50 mm beta-Glc 1-P and 50 mm glucose, glucosamine, N-acetyl glucosamine, mannose, xylose or l-fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product structures were determined by NMR and ESI-MS to be alpha-Glcp-(1 -> 4)-Glcp (maltose), alpha-Glcp-(1 -> 4)-GlcNp (maltosamine), alpha-Glcp-(1 -> 4)-GlcNAcp (N-acetyl maltosamine), alpha-Glcp-(1 -> 4)-Manp, alpha-Glcp-(1 -> 4)-Xylp and alpha-Glcp-(1 -> 4)- l-Fucp, the three latter being novel compounds. Modelling using L. brevis GH65 as the template and superimposition of acarbose from a complex with Thermoanaerobacterium thermosaccharolyticum GH15 glucoamylase suggested that loop 3 of MalP involved in substrate recognition blocked the binding of candidate acceptors larger than monosaccharides.
    Original languageEnglish
    JournalF E B S Journal
    Volume276
    Issue number24
    Pages (from-to)7353-7365
    ISSN1742-464X
    DOIs
    Publication statusPublished - 2009

    Keywords

    • reverse phosphorolysis
    • maltodextrin gene cluster
    • glycoside hydrolase family 65
    • beta-glucose 1-phosphate
    • alpha-glucosides

    Cite this

    @article{c75832dc21bf45cb8ad266fce97fe61a,
    title = "The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel alpha-glucosides through reverse phosphorolysis by maltose phosphorylase",
    abstract = "A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI-GalR family. Enzymatic properties are described for recombinant maltose phosphorylase (MalP) of glycoside hydrolase family 65 (GH65), which is encoded by malP (GenBank: AAV43670.1) of this gene cluster and produced in Escherichia coli. MalP catalyses phosphorolysis of maltose with inversion of the anomeric configuration releasing beta-glucose 1-phosphate (beta-Glc 1-P) and glucose. The broad specificity of the aglycone binding site was demonstrated by products formed in reverse phosphorolysis using various carbohydrate acceptor substrates and beta-Glc 1-P as the donor. MalP showed strong preference for monosaccharide acceptors with equatorial 3-OH and 4-OH, such as glucose and mannose, and also reacted with 2-deoxy glucosamine and 2-deoxy N-acetyl glucosamine. By contrast, none of the tested di- and trisaccharides served as acceptors. Disaccharide yields obtained from 50 mm beta-Glc 1-P and 50 mm glucose, glucosamine, N-acetyl glucosamine, mannose, xylose or l-fucose were 99, 80, 53, 93, 81 and 13{\%}, respectively. Product structures were determined by NMR and ESI-MS to be alpha-Glcp-(1 -> 4)-Glcp (maltose), alpha-Glcp-(1 -> 4)-GlcNp (maltosamine), alpha-Glcp-(1 -> 4)-GlcNAcp (N-acetyl maltosamine), alpha-Glcp-(1 -> 4)-Manp, alpha-Glcp-(1 -> 4)-Xylp and alpha-Glcp-(1 -> 4)- l-Fucp, the three latter being novel compounds. Modelling using L. brevis GH65 as the template and superimposition of acarbose from a complex with Thermoanaerobacterium thermosaccharolyticum GH15 glucoamylase suggested that loop 3 of MalP involved in substrate recognition blocked the binding of candidate acceptors larger than monosaccharides.",
    keywords = "reverse phosphorolysis, maltodextrin gene cluster, glycoside hydrolase family 65, beta-glucose 1-phosphate, alpha-glucosides",
    author = "Hiroyuki Nakai and Martin Baumann and B.O. Petersen and Y. Westphal and H. Schols and Adiphol Dilokpimol and {Abou Hachem}, Maher and S.J. Lahtinen and Duus, {Jens {\O}llgaard} and Birte Svensson",
    year = "2009",
    doi = "10.1111/j.1742-4658.2009.07445.x",
    language = "English",
    volume = "276",
    pages = "7353--7365",
    journal = "F E B S Journal",
    issn = "1742-464X",
    publisher = "Wiley-Blackwell",
    number = "24",

    }

    The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel alpha-glucosides through reverse phosphorolysis by maltose phosphorylase. / Nakai, Hiroyuki; Baumann, Martin; Petersen, B.O.; Westphal, Y.; Schols, H.; Dilokpimol, Adiphol; Abou Hachem, Maher; Lahtinen, S.J.; Duus, Jens Øllgaard; Svensson, Birte.

    In: F E B S Journal, Vol. 276, No. 24, 2009, p. 7353-7365.

    Research output: Contribution to journalJournal articleResearchpeer-review

    TY - JOUR

    T1 - The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel alpha-glucosides through reverse phosphorolysis by maltose phosphorylase

    AU - Nakai, Hiroyuki

    AU - Baumann, Martin

    AU - Petersen, B.O.

    AU - Westphal, Y.

    AU - Schols, H.

    AU - Dilokpimol, Adiphol

    AU - Abou Hachem, Maher

    AU - Lahtinen, S.J.

    AU - Duus, Jens Øllgaard

    AU - Svensson, Birte

    PY - 2009

    Y1 - 2009

    N2 - A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI-GalR family. Enzymatic properties are described for recombinant maltose phosphorylase (MalP) of glycoside hydrolase family 65 (GH65), which is encoded by malP (GenBank: AAV43670.1) of this gene cluster and produced in Escherichia coli. MalP catalyses phosphorolysis of maltose with inversion of the anomeric configuration releasing beta-glucose 1-phosphate (beta-Glc 1-P) and glucose. The broad specificity of the aglycone binding site was demonstrated by products formed in reverse phosphorolysis using various carbohydrate acceptor substrates and beta-Glc 1-P as the donor. MalP showed strong preference for monosaccharide acceptors with equatorial 3-OH and 4-OH, such as glucose and mannose, and also reacted with 2-deoxy glucosamine and 2-deoxy N-acetyl glucosamine. By contrast, none of the tested di- and trisaccharides served as acceptors. Disaccharide yields obtained from 50 mm beta-Glc 1-P and 50 mm glucose, glucosamine, N-acetyl glucosamine, mannose, xylose or l-fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product structures were determined by NMR and ESI-MS to be alpha-Glcp-(1 -> 4)-Glcp (maltose), alpha-Glcp-(1 -> 4)-GlcNp (maltosamine), alpha-Glcp-(1 -> 4)-GlcNAcp (N-acetyl maltosamine), alpha-Glcp-(1 -> 4)-Manp, alpha-Glcp-(1 -> 4)-Xylp and alpha-Glcp-(1 -> 4)- l-Fucp, the three latter being novel compounds. Modelling using L. brevis GH65 as the template and superimposition of acarbose from a complex with Thermoanaerobacterium thermosaccharolyticum GH15 glucoamylase suggested that loop 3 of MalP involved in substrate recognition blocked the binding of candidate acceptors larger than monosaccharides.

    AB - A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI-GalR family. Enzymatic properties are described for recombinant maltose phosphorylase (MalP) of glycoside hydrolase family 65 (GH65), which is encoded by malP (GenBank: AAV43670.1) of this gene cluster and produced in Escherichia coli. MalP catalyses phosphorolysis of maltose with inversion of the anomeric configuration releasing beta-glucose 1-phosphate (beta-Glc 1-P) and glucose. The broad specificity of the aglycone binding site was demonstrated by products formed in reverse phosphorolysis using various carbohydrate acceptor substrates and beta-Glc 1-P as the donor. MalP showed strong preference for monosaccharide acceptors with equatorial 3-OH and 4-OH, such as glucose and mannose, and also reacted with 2-deoxy glucosamine and 2-deoxy N-acetyl glucosamine. By contrast, none of the tested di- and trisaccharides served as acceptors. Disaccharide yields obtained from 50 mm beta-Glc 1-P and 50 mm glucose, glucosamine, N-acetyl glucosamine, mannose, xylose or l-fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product structures were determined by NMR and ESI-MS to be alpha-Glcp-(1 -> 4)-Glcp (maltose), alpha-Glcp-(1 -> 4)-GlcNp (maltosamine), alpha-Glcp-(1 -> 4)-GlcNAcp (N-acetyl maltosamine), alpha-Glcp-(1 -> 4)-Manp, alpha-Glcp-(1 -> 4)-Xylp and alpha-Glcp-(1 -> 4)- l-Fucp, the three latter being novel compounds. Modelling using L. brevis GH65 as the template and superimposition of acarbose from a complex with Thermoanaerobacterium thermosaccharolyticum GH15 glucoamylase suggested that loop 3 of MalP involved in substrate recognition blocked the binding of candidate acceptors larger than monosaccharides.

    KW - reverse phosphorolysis

    KW - maltodextrin gene cluster

    KW - glycoside hydrolase family 65

    KW - beta-glucose 1-phosphate

    KW - alpha-glucosides

    U2 - 10.1111/j.1742-4658.2009.07445.x

    DO - 10.1111/j.1742-4658.2009.07445.x

    M3 - Journal article

    VL - 276

    SP - 7353

    EP - 7365

    JO - F E B S Journal

    JF - F E B S Journal

    SN - 1742-464X

    IS - 24

    ER -