Alteration of the substrate specificity of L-amino acid ligase and selective synthesis of Met-Gly as a salt taste enhancer

Haruka Kino, Kuniki Kino

    Research output: Contribution to journalArticle

    5 Citations (Scopus)

    Abstract

    Dipeptides have unique physiological functions. This study focused on the salt-taste-enhancing dipeptide Met-Gly. BL00235, an L-amino acid ligase from Bacillus licheniformis NBRC12200, synthesizes Met-Gly as a major product as well as Met-Met as a by-product. To alter the substrate specificity of BL00235 and synthesize Met-Gly selectively, we chose to alter Pro85 residue based on the BL00235 crystal structure. We predicted that Met might be not recognized as a C-terminal substrate by occupying the space around C-terminal substrate. Pro85 was replaced with Phe, Tyr, and Trp, which have bulky aromatic side chains, by site-directed mutagenesis. These mutants lost the capacity to synthesize Met-Met, during the synthesis of Met-Gly. Furthermore, they did not synthesize Met-Met, even when methionine was used as a substrate. These results show that the amino acid residue at position 85 has a key role in C-terminal substrate specificity.

    Original languageEnglish
    Pages (from-to)1827-1832
    Number of pages6
    JournalBioscience, Biotechnology and Biochemistry
    Volume79
    Issue number11
    DOIs
    Publication statusPublished - 2015

    Fingerprint

    methionylglycine
    methionylmethionine
    Ligases
    Substrate Specificity
    Salts
    Amino Acids
    Dipeptides
    Substrates
    Site-Directed Mutagenesis
    Mutagenesis
    Methionine
    Bacilli
    Byproducts
    Crystal structure

    Keywords

    • Dipeptides
    • L-amino acid ligase
    • Salt taste enhancer
    • Site-directed mutagenesis

    ASJC Scopus subject areas

    • Biotechnology
    • Biochemistry
    • Molecular Biology
    • Applied Microbiology and Biotechnology
    • Analytical Chemistry
    • Organic Chemistry

    Cite this

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    abstract = "Dipeptides have unique physiological functions. This study focused on the salt-taste-enhancing dipeptide Met-Gly. BL00235, an L-amino acid ligase from Bacillus licheniformis NBRC12200, synthesizes Met-Gly as a major product as well as Met-Met as a by-product. To alter the substrate specificity of BL00235 and synthesize Met-Gly selectively, we chose to alter Pro85 residue based on the BL00235 crystal structure. We predicted that Met might be not recognized as a C-terminal substrate by occupying the space around C-terminal substrate. Pro85 was replaced with Phe, Tyr, and Trp, which have bulky aromatic side chains, by site-directed mutagenesis. These mutants lost the capacity to synthesize Met-Met, during the synthesis of Met-Gly. Furthermore, they did not synthesize Met-Met, even when methionine was used as a substrate. These results show that the amino acid residue at position 85 has a key role in C-terminal substrate specificity.",
    keywords = "Dipeptides, L-amino acid ligase, Salt taste enhancer, Site-directed mutagenesis",
    author = "Haruka Kino and Kuniki Kino",
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    AU - Kino, Haruka

    AU - Kino, Kuniki

    PY - 2015

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    N2 - Dipeptides have unique physiological functions. This study focused on the salt-taste-enhancing dipeptide Met-Gly. BL00235, an L-amino acid ligase from Bacillus licheniformis NBRC12200, synthesizes Met-Gly as a major product as well as Met-Met as a by-product. To alter the substrate specificity of BL00235 and synthesize Met-Gly selectively, we chose to alter Pro85 residue based on the BL00235 crystal structure. We predicted that Met might be not recognized as a C-terminal substrate by occupying the space around C-terminal substrate. Pro85 was replaced with Phe, Tyr, and Trp, which have bulky aromatic side chains, by site-directed mutagenesis. These mutants lost the capacity to synthesize Met-Met, during the synthesis of Met-Gly. Furthermore, they did not synthesize Met-Met, even when methionine was used as a substrate. These results show that the amino acid residue at position 85 has a key role in C-terminal substrate specificity.

    AB - Dipeptides have unique physiological functions. This study focused on the salt-taste-enhancing dipeptide Met-Gly. BL00235, an L-amino acid ligase from Bacillus licheniformis NBRC12200, synthesizes Met-Gly as a major product as well as Met-Met as a by-product. To alter the substrate specificity of BL00235 and synthesize Met-Gly selectively, we chose to alter Pro85 residue based on the BL00235 crystal structure. We predicted that Met might be not recognized as a C-terminal substrate by occupying the space around C-terminal substrate. Pro85 was replaced with Phe, Tyr, and Trp, which have bulky aromatic side chains, by site-directed mutagenesis. These mutants lost the capacity to synthesize Met-Met, during the synthesis of Met-Gly. Furthermore, they did not synthesize Met-Met, even when methionine was used as a substrate. These results show that the amino acid residue at position 85 has a key role in C-terminal substrate specificity.

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    KW - L-amino acid ligase

    KW - Salt taste enhancer

    KW - Site-directed mutagenesis

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