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

    15 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

    Keywords

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

    ASJC Scopus subject areas

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

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