The evolutionary appearance of non-cyanogenic hydroxynitrile glucosides in the Lotus genus is accompanied by the substrate specialization of paralogous beta-glucosidases resulting from a crucial amino acid substitution

Daniela Lai, Maher Abou Hachem, Fran Robson, Carl Erik Olsen, Trevor L. Wang, Birger Lindberg Møller, Adam M. Takos, Fred Rook

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    Lotus japonicus, like several other legumes, biosynthesizes the cyanogenic alpha-hydroxynitrile glucosides lot-australin and linamarin. Upon tissue disruption these compounds are hydrolysed by a specific beta-glucosidase, resulting in the release of hydrogen cyanide. Lotus japonicus also produces the non-cyanogenic gamma- and beta-hydroxynitrile glucosides rhodiocyanoside A and D using a biosynthetic pathway that branches off from lotaustralin biosynthesis. We previously established that BGD2 is the only beta-glucosidase responsible for cyanogenesis in leaves. Here we show that the paralogous BGD4 has the dominant physiological role in rhodiocyanoside degradation. Structural modelling, site-directed mutagenesis and activity assays establish that a glycine residue (G211) in the aglycone binding site of BGD2 is essential for its ability to hydrolyse the endogenous cyanogenic glucosides. The corresponding valine (V211) in BGD4 narrows the active site pocket, resulting in the exclusion of non-flat substrates such as lotaustralin and linamarin, but not of the more planar rhodiocyanosides. Rhodiocyanosides and the BGD4 gene only occur in L. japonicus and a few closely related species associated with the Lotus corniculatus clade within the Lotus genus. This suggests the evolutionary scenario that substrate specialization for rhodiocyanosides evolved from a promiscuous activity of a progenitor cyanogenic beta-glucosidase, resembling BGD2, and required no more than a single amino acid substitution.
    Original languageEnglish
    JournalPlant Journal
    Volume79
    Issue number2
    Pages (from-to)299-311
    Number of pages13
    ISSN0960-7412
    DOIs
    Publication statusPublished - 2014

    Keywords

    • enzyme activity
    • evolutionary scenario
    • genbank sequence data
    • Dicotyledones Angiospermae Spermatophyta Plantae (Angiosperms, Dicots, Plants, Spermatophytes, Vascular Plants) - Leguminosae [26260] Lotus japonicus species
    • Lotus japonicus BGD4 gene [Leguminosae] expression, transcription
    • australine 118396-02-4
    • beta-glucosidase 9001-22-3 EC 3.2.1.21
    • bete-hydroxynitrile glucosides rhodiocyanoside A signaling
    • bete-hydroxynitrile glucosides rhodiocyanoside D signaling
    • crucial amino acid
    • hydrogen cyanide 74-90-8
    • linamarin 554-35-8
    • valine 516-06-3
    • 03502, Genetics - General
    • 03504, Genetics - Plant
    • 10064, Biochemistry studies - Proteins, peptides and amino acids
    • 10802, Enzymes - General and comparative studies: coenzymes
    • 12002, Physiology - General
    • 51518, Plant physiology - Enzymes
    • 51526, Plant physiology - General and miscellaneous
    • Biochemistry and Molecular Biophysics
    • leaf
    • Chemical Coordination and Homeostasis
    • Enzymology
    • Molecular Genetics
    • PLANT
    • AGLYCONE SPECIFICITY
    • CRYSTAL-STRUCTURE
    • SESQUITERPENE SYNTHASES
    • O-METHYLTRANSFERASES
    • MAXIMUM-LIKELIHOOD
    • BIOSYNTHETIC GENES
    • REVERSE GENETICS
    • WHITE CLOVER
    • JAPONICUS
    • SORGHUM
    • Lotus japonicus
    • beta-glucosidase
    • enzyme evolution
    • cyanogenesis
    • hydroxynitrile glucosides
    • rhodiocyanosides
    • Lotus burttii
    • Lotus krylovii
    • Lotus filicaulis
    • Lotus corniculatus
    • β–glucosidase

    Cite this

    @article{1d4ba0da754142fcb2c97e6694c0b199,
    title = "The evolutionary appearance of non-cyanogenic hydroxynitrile glucosides in the Lotus genus is accompanied by the substrate specialization of paralogous beta-glucosidases resulting from a crucial amino acid substitution",
    abstract = "Lotus japonicus, like several other legumes, biosynthesizes the cyanogenic alpha-hydroxynitrile glucosides lot-australin and linamarin. Upon tissue disruption these compounds are hydrolysed by a specific beta-glucosidase, resulting in the release of hydrogen cyanide. Lotus japonicus also produces the non-cyanogenic gamma- and beta-hydroxynitrile glucosides rhodiocyanoside A and D using a biosynthetic pathway that branches off from lotaustralin biosynthesis. We previously established that BGD2 is the only beta-glucosidase responsible for cyanogenesis in leaves. Here we show that the paralogous BGD4 has the dominant physiological role in rhodiocyanoside degradation. Structural modelling, site-directed mutagenesis and activity assays establish that a glycine residue (G211) in the aglycone binding site of BGD2 is essential for its ability to hydrolyse the endogenous cyanogenic glucosides. The corresponding valine (V211) in BGD4 narrows the active site pocket, resulting in the exclusion of non-flat substrates such as lotaustralin and linamarin, but not of the more planar rhodiocyanosides. Rhodiocyanosides and the BGD4 gene only occur in L. japonicus and a few closely related species associated with the Lotus corniculatus clade within the Lotus genus. This suggests the evolutionary scenario that substrate specialization for rhodiocyanosides evolved from a promiscuous activity of a progenitor cyanogenic beta-glucosidase, resembling BGD2, and required no more than a single amino acid substitution.",
    keywords = "enzyme activity, evolutionary scenario, genbank sequence data, Dicotyledones Angiospermae Spermatophyta Plantae (Angiosperms, Dicots, Plants, Spermatophytes, Vascular Plants) - Leguminosae [26260] Lotus japonicus species, Lotus japonicus BGD4 gene [Leguminosae] expression, transcription, australine 118396-02-4, beta-glucosidase 9001-22-3 EC 3.2.1.21, bete-hydroxynitrile glucosides rhodiocyanoside A signaling, bete-hydroxynitrile glucosides rhodiocyanoside D signaling, crucial amino acid, hydrogen cyanide 74-90-8, linamarin 554-35-8, valine 516-06-3, 03502, Genetics - General, 03504, Genetics - Plant, 10064, Biochemistry studies - Proteins, peptides and amino acids, 10802, Enzymes - General and comparative studies: coenzymes, 12002, Physiology - General, 51518, Plant physiology - Enzymes, 51526, Plant physiology - General and miscellaneous, Biochemistry and Molecular Biophysics, leaf, Chemical Coordination and Homeostasis, Enzymology, Molecular Genetics, PLANT, AGLYCONE SPECIFICITY, CRYSTAL-STRUCTURE, SESQUITERPENE SYNTHASES, O-METHYLTRANSFERASES, MAXIMUM-LIKELIHOOD, BIOSYNTHETIC GENES, REVERSE GENETICS, WHITE CLOVER, JAPONICUS, SORGHUM, Lotus japonicus, beta-glucosidase, enzyme evolution, cyanogenesis, hydroxynitrile glucosides, rhodiocyanosides, Lotus burttii, Lotus krylovii, Lotus filicaulis, Lotus corniculatus, β–glucosidase",
    author = "Daniela Lai and {Abou Hachem}, Maher and Fran Robson and Olsen, {Carl Erik} and Wang, {Trevor L.} and M{\o}ller, {Birger Lindberg} and Takos, {Adam M.} and Fred Rook",
    year = "2014",
    doi = "10.1111/tpj.12561",
    language = "English",
    volume = "79",
    pages = "299--311",
    journal = "Plant Journal",
    issn = "0960-7412",
    publisher = "Wiley-Blackwell",
    number = "2",

    }

    The evolutionary appearance of non-cyanogenic hydroxynitrile glucosides in the Lotus genus is accompanied by the substrate specialization of paralogous beta-glucosidases resulting from a crucial amino acid substitution. / Lai, Daniela; Abou Hachem, Maher ; Robson, Fran; Olsen, Carl Erik; Wang, Trevor L.; Møller, Birger Lindberg; Takos, Adam M.; Rook, Fred.

    In: Plant Journal, Vol. 79, No. 2, 2014, p. 299-311.

    Research output: Contribution to journalJournal articleResearchpeer-review

    TY - JOUR

    T1 - The evolutionary appearance of non-cyanogenic hydroxynitrile glucosides in the Lotus genus is accompanied by the substrate specialization of paralogous beta-glucosidases resulting from a crucial amino acid substitution

    AU - Lai, Daniela

    AU - Abou Hachem, Maher

    AU - Robson, Fran

    AU - Olsen, Carl Erik

    AU - Wang, Trevor L.

    AU - Møller, Birger Lindberg

    AU - Takos, Adam M.

    AU - Rook, Fred

    PY - 2014

    Y1 - 2014

    N2 - Lotus japonicus, like several other legumes, biosynthesizes the cyanogenic alpha-hydroxynitrile glucosides lot-australin and linamarin. Upon tissue disruption these compounds are hydrolysed by a specific beta-glucosidase, resulting in the release of hydrogen cyanide. Lotus japonicus also produces the non-cyanogenic gamma- and beta-hydroxynitrile glucosides rhodiocyanoside A and D using a biosynthetic pathway that branches off from lotaustralin biosynthesis. We previously established that BGD2 is the only beta-glucosidase responsible for cyanogenesis in leaves. Here we show that the paralogous BGD4 has the dominant physiological role in rhodiocyanoside degradation. Structural modelling, site-directed mutagenesis and activity assays establish that a glycine residue (G211) in the aglycone binding site of BGD2 is essential for its ability to hydrolyse the endogenous cyanogenic glucosides. The corresponding valine (V211) in BGD4 narrows the active site pocket, resulting in the exclusion of non-flat substrates such as lotaustralin and linamarin, but not of the more planar rhodiocyanosides. Rhodiocyanosides and the BGD4 gene only occur in L. japonicus and a few closely related species associated with the Lotus corniculatus clade within the Lotus genus. This suggests the evolutionary scenario that substrate specialization for rhodiocyanosides evolved from a promiscuous activity of a progenitor cyanogenic beta-glucosidase, resembling BGD2, and required no more than a single amino acid substitution.

    AB - Lotus japonicus, like several other legumes, biosynthesizes the cyanogenic alpha-hydroxynitrile glucosides lot-australin and linamarin. Upon tissue disruption these compounds are hydrolysed by a specific beta-glucosidase, resulting in the release of hydrogen cyanide. Lotus japonicus also produces the non-cyanogenic gamma- and beta-hydroxynitrile glucosides rhodiocyanoside A and D using a biosynthetic pathway that branches off from lotaustralin biosynthesis. We previously established that BGD2 is the only beta-glucosidase responsible for cyanogenesis in leaves. Here we show that the paralogous BGD4 has the dominant physiological role in rhodiocyanoside degradation. Structural modelling, site-directed mutagenesis and activity assays establish that a glycine residue (G211) in the aglycone binding site of BGD2 is essential for its ability to hydrolyse the endogenous cyanogenic glucosides. The corresponding valine (V211) in BGD4 narrows the active site pocket, resulting in the exclusion of non-flat substrates such as lotaustralin and linamarin, but not of the more planar rhodiocyanosides. Rhodiocyanosides and the BGD4 gene only occur in L. japonicus and a few closely related species associated with the Lotus corniculatus clade within the Lotus genus. This suggests the evolutionary scenario that substrate specialization for rhodiocyanosides evolved from a promiscuous activity of a progenitor cyanogenic beta-glucosidase, resembling BGD2, and required no more than a single amino acid substitution.

    KW - enzyme activity

    KW - evolutionary scenario

    KW - genbank sequence data

    KW - Dicotyledones Angiospermae Spermatophyta Plantae (Angiosperms, Dicots, Plants, Spermatophytes, Vascular Plants) - Leguminosae [26260] Lotus japonicus species

    KW - Lotus japonicus BGD4 gene [Leguminosae] expression, transcription

    KW - australine 118396-02-4

    KW - beta-glucosidase 9001-22-3 EC 3.2.1.21

    KW - bete-hydroxynitrile glucosides rhodiocyanoside A signaling

    KW - bete-hydroxynitrile glucosides rhodiocyanoside D signaling

    KW - crucial amino acid

    KW - hydrogen cyanide 74-90-8

    KW - linamarin 554-35-8

    KW - valine 516-06-3

    KW - 03502, Genetics - General

    KW - 03504, Genetics - Plant

    KW - 10064, Biochemistry studies - Proteins, peptides and amino acids

    KW - 10802, Enzymes - General and comparative studies: coenzymes

    KW - 12002, Physiology - General

    KW - 51518, Plant physiology - Enzymes

    KW - 51526, Plant physiology - General and miscellaneous

    KW - Biochemistry and Molecular Biophysics

    KW - leaf

    KW - Chemical Coordination and Homeostasis

    KW - Enzymology

    KW - Molecular Genetics

    KW - PLANT

    KW - AGLYCONE SPECIFICITY

    KW - CRYSTAL-STRUCTURE

    KW - SESQUITERPENE SYNTHASES

    KW - O-METHYLTRANSFERASES

    KW - MAXIMUM-LIKELIHOOD

    KW - BIOSYNTHETIC GENES

    KW - REVERSE GENETICS

    KW - WHITE CLOVER

    KW - JAPONICUS

    KW - SORGHUM

    KW - Lotus japonicus

    KW - beta-glucosidase

    KW - enzyme evolution

    KW - cyanogenesis

    KW - hydroxynitrile glucosides

    KW - rhodiocyanosides

    KW - Lotus burttii

    KW - Lotus krylovii

    KW - Lotus filicaulis

    KW - Lotus corniculatus

    KW - β–glucosidase

    U2 - 10.1111/tpj.12561

    DO - 10.1111/tpj.12561

    M3 - Journal article

    VL - 79

    SP - 299

    EP - 311

    JO - Plant Journal

    JF - Plant Journal

    SN - 0960-7412

    IS - 2

    ER -