Refined regio- and stereoselective hydroxylation of l -pipecolic acid by protein engineering of l -proline cis -4-hydroxylase based on the X-ray crystal structure

Kento Koketsu, Yasuhito Shomura, Kei Moriwaki, Mikiro Hayashi, Satoshi Mitsuhashi, Ryotaro Hara, Kuniki Kino, Yoshiki Higuchi

    Research output: Contribution to journalArticle

    11 Citations (Scopus)

    Abstract

    Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Proline hydroxylases are representative members of the nonheme Fe<sup>2+</sup>/α-ketoglutarate-dependent dioxygenase family. These enzymes catalyze the conversion of l-proline into hydroxy-l-prolines (Hyps). l-Proline cis-4-hydroxylases (cis-P4Hs) from Sinorhizobium meliloti and Mesorhizobium loti catalyze the hydroxylation of l-proline, generating cis-4-hydroxy-l-proline, as well as the hydroxylation of l-pipecolic acid (l-Pip), generating two regioisomers, cis-5-Hypip and cis-3-Hypip. To selectively produce cis-5-Hypip without simultaneous production of two isomers, protein engineering of cis-P4Hs is required. We therefore carried out protein engineering of cis-P4H to facilitate the conversion of the majority of l-Pip into the cis-5-Hypip isomer. We first solved the X-ray crystal structure of cis-P4H in complex with each of l-Pro and l-Pip. Then, we conducted three rounds of directed evolution and successfully created a cis-P4H triple mutant, V97F/V95W/E114G, demonstrating the desired regioselectivity toward cis-5-Hypip.

    Original languageEnglish
    Pages (from-to)383-392
    Number of pages10
    JournalACS Synthetic Biology
    Volume4
    Issue number4
    DOIs
    Publication statusPublished - 2015 Apr 17

    Fingerprint

    Protein Engineering
    Hydroxylation
    Mixed Function Oxygenases
    Proline
    Hydroxyproline
    Crystal structure
    X-Rays
    Proteins
    X rays
    Isomers
    Acids
    Prolyl Hydroxylases
    Dioxygenases
    Regioselectivity
    Mesorhizobium
    Sinorhizobium meliloti
    Enzymes
    pipecolic acid

    Keywords

    • hydroxy- l -pipecolic acid
    • hydroxylation
    • hydroxyproline
    • proline hydroxylase
    • protein engineering

    ASJC Scopus subject areas

    • Biochemistry, Genetics and Molecular Biology (miscellaneous)
    • Biomedical Engineering

    Cite this

    Refined regio- and stereoselective hydroxylation of l -pipecolic acid by protein engineering of l -proline cis -4-hydroxylase based on the X-ray crystal structure. / Koketsu, Kento; Shomura, Yasuhito; Moriwaki, Kei; Hayashi, Mikiro; Mitsuhashi, Satoshi; Hara, Ryotaro; Kino, Kuniki; Higuchi, Yoshiki.

    In: ACS Synthetic Biology, Vol. 4, No. 4, 17.04.2015, p. 383-392.

    Research output: Contribution to journalArticle

    Koketsu, Kento ; Shomura, Yasuhito ; Moriwaki, Kei ; Hayashi, Mikiro ; Mitsuhashi, Satoshi ; Hara, Ryotaro ; Kino, Kuniki ; Higuchi, Yoshiki. / Refined regio- and stereoselective hydroxylation of l -pipecolic acid by protein engineering of l -proline cis -4-hydroxylase based on the X-ray crystal structure. In: ACS Synthetic Biology. 2015 ; Vol. 4, No. 4. pp. 383-392.
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    AU - Koketsu, Kento

    AU - Shomura, Yasuhito

    AU - Moriwaki, Kei

    AU - Hayashi, Mikiro

    AU - Mitsuhashi, Satoshi

    AU - Hara, Ryotaro

    AU - Kino, Kuniki

    AU - Higuchi, Yoshiki

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    AB - Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Proline hydroxylases are representative members of the nonheme Fe2+/α-ketoglutarate-dependent dioxygenase family. These enzymes catalyze the conversion of l-proline into hydroxy-l-prolines (Hyps). l-Proline cis-4-hydroxylases (cis-P4Hs) from Sinorhizobium meliloti and Mesorhizobium loti catalyze the hydroxylation of l-proline, generating cis-4-hydroxy-l-proline, as well as the hydroxylation of l-pipecolic acid (l-Pip), generating two regioisomers, cis-5-Hypip and cis-3-Hypip. To selectively produce cis-5-Hypip without simultaneous production of two isomers, protein engineering of cis-P4Hs is required. We therefore carried out protein engineering of cis-P4H to facilitate the conversion of the majority of l-Pip into the cis-5-Hypip isomer. We first solved the X-ray crystal structure of cis-P4H in complex with each of l-Pro and l-Pip. Then, we conducted three rounds of directed evolution and successfully created a cis-P4H triple mutant, V97F/V95W/E114G, demonstrating the desired regioselectivity toward cis-5-Hypip.

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