Crystal Structure of α-Galactosidase from Lactobacillus acidophilus NCFM: Insight into Tetramer Formation and Substrate Binding

Folmer Fredslund, Maher Abou Hachem, Rene Jonsgaard Larsen, Pernille Gerd Sørensen, Pedro M. Coutinho, Leila Lo Leggio, Birte Svensson

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    Lactobacillus acidophilus NCFM is a probiotic bacterium known for its beneficial effects on human health. The importance of α-galactosidases (α-Gals) for growth of probiotic organisms on oligosaccharides of the raffinose family present in many foods is increasingly recognized. Here, the crystal structure of α-Gal from L. acidophilus NCFM (LaMel36A) of glycoside hydrolase (GH) family 36 (GH36) is determined by single-wavelength anomalous dispersion. In addition, a 1.58-Å-resolution crystallographic complex with α-d-galactose at substrate binding subsite −1 was determined. LaMel36A has a large N-terminal twisted β-sandwich domain, connected by a long α-helix to the catalytic (β/α)8-barrel domain, and a C-terminal β-sheet domain. Four identical monomers form a tightly packed tetramer where three monomers contribute to the structural integrity of the active site in each monomer. Structural comparison of LaMel36A with the monomeric Thermotoga maritima α-Gal (TmGal36A) reveals that O2 of α-d-galactose in LaMel36A interacts with a backbone nitrogen in a glycine-rich loop of the catalytic domain, whereas the corresponding atom in TmGal36A is from a tryptophan side chain belonging to the N-terminal domain. Thus, two distinctly different structural motifs participate in substrate recognition. The tetrameric LaMel36A furthermore has a much deeper active site than the monomeric TmGal36A, which possibly modulates substrate specificity. Sequence analysis of GH36, inspired by the observed structural differences, results in four distinct subgroups having clearly different active-site sequence motifs. This novel subdivision incorporates functional and architectural features and may aid further biochemical and structural analyses within GH36.
    Original languageEnglish
    JournalJournal of Molecular Biology
    Volume412
    Issue number3
    Pages (from-to)466-480
    ISSN0022-2836
    DOIs
    Publication statusPublished - 2011

    Keywords

    • Probiotics
    • Glycoside hydrolase
    • Crystal structure
    • Family 36
    • Substrate specificity
    • Quaternary structure

    Cite this

    @article{2779af92ae0c481eafea6176eb2380fc,
    title = "Crystal Structure of α-Galactosidase from Lactobacillus acidophilus NCFM: Insight into Tetramer Formation and Substrate Binding",
    abstract = "Lactobacillus acidophilus NCFM is a probiotic bacterium known for its beneficial effects on human health. The importance of α-galactosidases (α-Gals) for growth of probiotic organisms on oligosaccharides of the raffinose family present in many foods is increasingly recognized. Here, the crystal structure of α-Gal from L. acidophilus NCFM (LaMel36A) of glycoside hydrolase (GH) family 36 (GH36) is determined by single-wavelength anomalous dispersion. In addition, a 1.58-{\AA}-resolution crystallographic complex with α-d-galactose at substrate binding subsite −1 was determined. LaMel36A has a large N-terminal twisted β-sandwich domain, connected by a long α-helix to the catalytic (β/α)8-barrel domain, and a C-terminal β-sheet domain. Four identical monomers form a tightly packed tetramer where three monomers contribute to the structural integrity of the active site in each monomer. Structural comparison of LaMel36A with the monomeric Thermotoga maritima α-Gal (TmGal36A) reveals that O2 of α-d-galactose in LaMel36A interacts with a backbone nitrogen in a glycine-rich loop of the catalytic domain, whereas the corresponding atom in TmGal36A is from a tryptophan side chain belonging to the N-terminal domain. Thus, two distinctly different structural motifs participate in substrate recognition. The tetrameric LaMel36A furthermore has a much deeper active site than the monomeric TmGal36A, which possibly modulates substrate specificity. Sequence analysis of GH36, inspired by the observed structural differences, results in four distinct subgroups having clearly different active-site sequence motifs. This novel subdivision incorporates functional and architectural features and may aid further biochemical and structural analyses within GH36.",
    keywords = "Probiotics, Glycoside hydrolase, Crystal structure, Family 36, Substrate specificity, Quaternary structure",
    author = "Folmer Fredslund and {Abou Hachem}, Maher and Larsen, {Rene Jonsgaard} and S{\o}rensen, {Pernille Gerd} and Coutinho, {Pedro M.} and {Lo Leggio}, Leila and Birte Svensson",
    year = "2011",
    doi = "10.1016/j.jmb.2011.07.057",
    language = "English",
    volume = "412",
    pages = "466--480",
    journal = "Journal of Molecular Biology",
    issn = "0022-2836",
    publisher = "Academic Press",
    number = "3",

    }

    Crystal Structure of α-Galactosidase from Lactobacillus acidophilus NCFM: Insight into Tetramer Formation and Substrate Binding. / Fredslund, Folmer; Abou Hachem, Maher; Larsen, Rene Jonsgaard; Sørensen, Pernille Gerd; Coutinho, Pedro M.; Lo Leggio, Leila; Svensson, Birte.

    In: Journal of Molecular Biology, Vol. 412, No. 3, 2011, p. 466-480.

    Research output: Contribution to journalJournal articleResearchpeer-review

    TY - JOUR

    T1 - Crystal Structure of α-Galactosidase from Lactobacillus acidophilus NCFM: Insight into Tetramer Formation and Substrate Binding

    AU - Fredslund, Folmer

    AU - Abou Hachem, Maher

    AU - Larsen, Rene Jonsgaard

    AU - Sørensen, Pernille Gerd

    AU - Coutinho, Pedro M.

    AU - Lo Leggio, Leila

    AU - Svensson, Birte

    PY - 2011

    Y1 - 2011

    N2 - Lactobacillus acidophilus NCFM is a probiotic bacterium known for its beneficial effects on human health. The importance of α-galactosidases (α-Gals) for growth of probiotic organisms on oligosaccharides of the raffinose family present in many foods is increasingly recognized. Here, the crystal structure of α-Gal from L. acidophilus NCFM (LaMel36A) of glycoside hydrolase (GH) family 36 (GH36) is determined by single-wavelength anomalous dispersion. In addition, a 1.58-Å-resolution crystallographic complex with α-d-galactose at substrate binding subsite −1 was determined. LaMel36A has a large N-terminal twisted β-sandwich domain, connected by a long α-helix to the catalytic (β/α)8-barrel domain, and a C-terminal β-sheet domain. Four identical monomers form a tightly packed tetramer where three monomers contribute to the structural integrity of the active site in each monomer. Structural comparison of LaMel36A with the monomeric Thermotoga maritima α-Gal (TmGal36A) reveals that O2 of α-d-galactose in LaMel36A interacts with a backbone nitrogen in a glycine-rich loop of the catalytic domain, whereas the corresponding atom in TmGal36A is from a tryptophan side chain belonging to the N-terminal domain. Thus, two distinctly different structural motifs participate in substrate recognition. The tetrameric LaMel36A furthermore has a much deeper active site than the monomeric TmGal36A, which possibly modulates substrate specificity. Sequence analysis of GH36, inspired by the observed structural differences, results in four distinct subgroups having clearly different active-site sequence motifs. This novel subdivision incorporates functional and architectural features and may aid further biochemical and structural analyses within GH36.

    AB - Lactobacillus acidophilus NCFM is a probiotic bacterium known for its beneficial effects on human health. The importance of α-galactosidases (α-Gals) for growth of probiotic organisms on oligosaccharides of the raffinose family present in many foods is increasingly recognized. Here, the crystal structure of α-Gal from L. acidophilus NCFM (LaMel36A) of glycoside hydrolase (GH) family 36 (GH36) is determined by single-wavelength anomalous dispersion. In addition, a 1.58-Å-resolution crystallographic complex with α-d-galactose at substrate binding subsite −1 was determined. LaMel36A has a large N-terminal twisted β-sandwich domain, connected by a long α-helix to the catalytic (β/α)8-barrel domain, and a C-terminal β-sheet domain. Four identical monomers form a tightly packed tetramer where three monomers contribute to the structural integrity of the active site in each monomer. Structural comparison of LaMel36A with the monomeric Thermotoga maritima α-Gal (TmGal36A) reveals that O2 of α-d-galactose in LaMel36A interacts with a backbone nitrogen in a glycine-rich loop of the catalytic domain, whereas the corresponding atom in TmGal36A is from a tryptophan side chain belonging to the N-terminal domain. Thus, two distinctly different structural motifs participate in substrate recognition. The tetrameric LaMel36A furthermore has a much deeper active site than the monomeric TmGal36A, which possibly modulates substrate specificity. Sequence analysis of GH36, inspired by the observed structural differences, results in four distinct subgroups having clearly different active-site sequence motifs. This novel subdivision incorporates functional and architectural features and may aid further biochemical and structural analyses within GH36.

    KW - Probiotics

    KW - Glycoside hydrolase

    KW - Crystal structure

    KW - Family 36

    KW - Substrate specificity

    KW - Quaternary structure

    U2 - 10.1016/j.jmb.2011.07.057

    DO - 10.1016/j.jmb.2011.07.057

    M3 - Journal article

    VL - 412

    SP - 466

    EP - 480

    JO - Journal of Molecular Biology

    JF - Journal of Molecular Biology

    SN - 0022-2836

    IS - 3

    ER -