Enhancement of l-tryptophan 5-hydroxylation activity by structure-based modification of l-phenylalanine 4-hydroxylase from Chromobacterium violaceum

Kuniki Kino, Ryotaro Hara, Ai Nozawa

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    6 Citations (Scopus)

    Abstract

    The objective of this study was to enhance l-tryptophan hydroxylation activity of l-phenylalanine 4-hydroxylase. It had been known that l-phenylalanine 4-hydroxylase from Chromobacterium violaceum could convert l-tryptophan to 5-hydroxy-l-tryptophan and l-phenylalanine to l-tyrosine; however, the activity for l-tryptophan was extremely low compared to l-phenylalanine activity levels. We used the information on the crystal structures of aromatic amino acid hydroxylases to generate C. violaceum l-phenylalanine 4-hydroxylase with high l-tryptophan hydroxylating activity. In silico structural modeling analysis suggested that hydrophobic and/or stacking interactions with the substrate and cofactor at L101 and W180 in C. violaceum l-phenylalanine 4-hydroxylase would increase hydroxylation activity. Based on this hypothesis, we introduced a saturation mutagenesis towards these sites followed by the evaluation of 5-hydroxy-l-tryptophan productivity using a modified Gibbs assay. Three and nine positive mutants were obtained from the L101 and W180 mutant libraries, respectively. Among the mutants, L101Y and W180F showed the highest l-tryptophan hydroxylation activity at the respective residues. Steady-state kinetic analysis revealed that kcat values for l-tryptophan hydroxylation were increased from 0.40 (wild-type) to 1.02 (L101Y) and 0.51 s- 1 (W180F). In addition, the double mutant (L101Y-W180F) displayed higher l-tryptophan hydroxylation activity than the wild-type and the W180F and L101Y mutants. The kcat value of L101Y-W180F increased to 2.08 s- 1, showing a 5.2-fold increase compared to wild-type enzyme levels.

    Original languageEnglish
    Pages (from-to)184-189
    Number of pages6
    JournalJournal of Bioscience and Bioengineering
    Volume108
    Issue number3
    DOIs
    Publication statusPublished - 2009 Sep

    Fingerprint

    Chromobacterium
    Phenylalanine Hydroxylase
    Hydroxylation
    Tryptophan
    5-Hydroxytryptophan
    Phenylalanine
    Mutagenesis
    Carboxylic acids
    Aromatic Amino Acids
    Amino acids
    Assays
    Enzymes
    Crystal structure
    Productivity
    Mixed Function Oxygenases
    Computer Simulation
    Tyrosine
    Kinetics
    Substrates

    Keywords

    • 5-Hydroxy-l-tryptophan
    • Aromatic amino acid hydroxylase
    • Chromobacterium violaceum
    • l-Phenylalanine 4-hydroxylase
    • Saturation mutagenesis

    ASJC Scopus subject areas

    • Biotechnology
    • Applied Microbiology and Biotechnology
    • Bioengineering

    Cite this

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    title = "Enhancement of l-tryptophan 5-hydroxylation activity by structure-based modification of l-phenylalanine 4-hydroxylase from Chromobacterium violaceum",
    abstract = "The objective of this study was to enhance l-tryptophan hydroxylation activity of l-phenylalanine 4-hydroxylase. It had been known that l-phenylalanine 4-hydroxylase from Chromobacterium violaceum could convert l-tryptophan to 5-hydroxy-l-tryptophan and l-phenylalanine to l-tyrosine; however, the activity for l-tryptophan was extremely low compared to l-phenylalanine activity levels. We used the information on the crystal structures of aromatic amino acid hydroxylases to generate C. violaceum l-phenylalanine 4-hydroxylase with high l-tryptophan hydroxylating activity. In silico structural modeling analysis suggested that hydrophobic and/or stacking interactions with the substrate and cofactor at L101 and W180 in C. violaceum l-phenylalanine 4-hydroxylase would increase hydroxylation activity. Based on this hypothesis, we introduced a saturation mutagenesis towards these sites followed by the evaluation of 5-hydroxy-l-tryptophan productivity using a modified Gibbs assay. Three and nine positive mutants were obtained from the L101 and W180 mutant libraries, respectively. Among the mutants, L101Y and W180F showed the highest l-tryptophan hydroxylation activity at the respective residues. Steady-state kinetic analysis revealed that kcat values for l-tryptophan hydroxylation were increased from 0.40 (wild-type) to 1.02 (L101Y) and 0.51 s- 1 (W180F). In addition, the double mutant (L101Y-W180F) displayed higher l-tryptophan hydroxylation activity than the wild-type and the W180F and L101Y mutants. The kcat value of L101Y-W180F increased to 2.08 s- 1, showing a 5.2-fold increase compared to wild-type enzyme levels.",
    keywords = "5-Hydroxy-l-tryptophan, Aromatic amino acid hydroxylase, Chromobacterium violaceum, l-Phenylalanine 4-hydroxylase, Saturation mutagenesis",
    author = "Kuniki Kino and Ryotaro Hara and Ai Nozawa",
    year = "2009",
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    language = "English",
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    T1 - Enhancement of l-tryptophan 5-hydroxylation activity by structure-based modification of l-phenylalanine 4-hydroxylase from Chromobacterium violaceum

    AU - Kino, Kuniki

    AU - Hara, Ryotaro

    AU - Nozawa, Ai

    PY - 2009/9

    Y1 - 2009/9

    N2 - The objective of this study was to enhance l-tryptophan hydroxylation activity of l-phenylalanine 4-hydroxylase. It had been known that l-phenylalanine 4-hydroxylase from Chromobacterium violaceum could convert l-tryptophan to 5-hydroxy-l-tryptophan and l-phenylalanine to l-tyrosine; however, the activity for l-tryptophan was extremely low compared to l-phenylalanine activity levels. We used the information on the crystal structures of aromatic amino acid hydroxylases to generate C. violaceum l-phenylalanine 4-hydroxylase with high l-tryptophan hydroxylating activity. In silico structural modeling analysis suggested that hydrophobic and/or stacking interactions with the substrate and cofactor at L101 and W180 in C. violaceum l-phenylalanine 4-hydroxylase would increase hydroxylation activity. Based on this hypothesis, we introduced a saturation mutagenesis towards these sites followed by the evaluation of 5-hydroxy-l-tryptophan productivity using a modified Gibbs assay. Three and nine positive mutants were obtained from the L101 and W180 mutant libraries, respectively. Among the mutants, L101Y and W180F showed the highest l-tryptophan hydroxylation activity at the respective residues. Steady-state kinetic analysis revealed that kcat values for l-tryptophan hydroxylation were increased from 0.40 (wild-type) to 1.02 (L101Y) and 0.51 s- 1 (W180F). In addition, the double mutant (L101Y-W180F) displayed higher l-tryptophan hydroxylation activity than the wild-type and the W180F and L101Y mutants. The kcat value of L101Y-W180F increased to 2.08 s- 1, showing a 5.2-fold increase compared to wild-type enzyme levels.

    AB - The objective of this study was to enhance l-tryptophan hydroxylation activity of l-phenylalanine 4-hydroxylase. It had been known that l-phenylalanine 4-hydroxylase from Chromobacterium violaceum could convert l-tryptophan to 5-hydroxy-l-tryptophan and l-phenylalanine to l-tyrosine; however, the activity for l-tryptophan was extremely low compared to l-phenylalanine activity levels. We used the information on the crystal structures of aromatic amino acid hydroxylases to generate C. violaceum l-phenylalanine 4-hydroxylase with high l-tryptophan hydroxylating activity. In silico structural modeling analysis suggested that hydrophobic and/or stacking interactions with the substrate and cofactor at L101 and W180 in C. violaceum l-phenylalanine 4-hydroxylase would increase hydroxylation activity. Based on this hypothesis, we introduced a saturation mutagenesis towards these sites followed by the evaluation of 5-hydroxy-l-tryptophan productivity using a modified Gibbs assay. Three and nine positive mutants were obtained from the L101 and W180 mutant libraries, respectively. Among the mutants, L101Y and W180F showed the highest l-tryptophan hydroxylation activity at the respective residues. Steady-state kinetic analysis revealed that kcat values for l-tryptophan hydroxylation were increased from 0.40 (wild-type) to 1.02 (L101Y) and 0.51 s- 1 (W180F). In addition, the double mutant (L101Y-W180F) displayed higher l-tryptophan hydroxylation activity than the wild-type and the W180F and L101Y mutants. The kcat value of L101Y-W180F increased to 2.08 s- 1, showing a 5.2-fold increase compared to wild-type enzyme levels.

    KW - 5-Hydroxy-l-tryptophan

    KW - Aromatic amino acid hydroxylase

    KW - Chromobacterium violaceum

    KW - l-Phenylalanine 4-hydroxylase

    KW - Saturation mutagenesis

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