Development of a multi-enzymatic cascade reaction for the synthesis of trans-3-hydroxy-l-proline from l-arginine

Ryotaro Hara, Saki Kitatsuji, Kai Yamagata, Kuniki Kino

    Research output: Contribution to journalArticle

    4 Citations (Scopus)

    Abstract

    Naturally occurring l-hydroxyproline in its four regio- and stereoisomeric forms has been explored as a possible precursor for pharmaceutical agents, yet the selective synthesis of trans-3-hydroxy-l-proline has not been achieved. Our aim was to develop a novel biocatalytic asymmetric method for the synthesis of trans-3-hydroxy-l-proline. So far, we focused on the rhizobial arginine catabolic pathway: arginase and ornithine cyclodeaminase are involved in l-arginine degradation to l-proline via l-ornithine. We hypothesized that trans-3-hydroxy-l-proline should be synthesized if arginase and ornithine cyclodeaminase act on (2S,3S)-3-hydroxyarginine and (2S,3S)-3-hydroxyornithine, respectively. To test this hypothesis, we cloned the genes of l-arginine 3-hydroxylase, arginase, and ornithine cyclodeaminase and overexpressed them in Escherichia coli, with subsequent enzyme purification. After characterization and optimization of each enzyme, a three-step procedure involving l-arginine 3-hydroxylase, arginase, and ornithine cyclodeaminase (in this order) was performed using l-arginine as a starting substrate. At the second step of the procedure, putative hydroxyornithine was formed quantitatively by arginase from (2S,3S)-3-hydroxyarginine. Nuclear magnetic resonance and chiral high-performance liquid chromatography analyses revealed that the absolute configuration of this compound was (2S,3S)-3-hydroxyornithine. In the last step of the procedure, trans-3-hydroxy-l-proline was synthesized selectively by ornithine cyclodeaminase from (2S,3S)-3-hydroxyornithine. Thus, we successfully developed a novel synthetic route, comprised of three reactions, to convert l-arginine to trans-3-hydroxy-l-proline. The excellent selectivity makes this procedure simpler and more efficient than conventional chemical synthesis.

    Original languageEnglish
    Pages (from-to)243-253
    Number of pages11
    JournalApplied Microbiology and Biotechnology
    Volume100
    Issue number1
    DOIs
    Publication statusPublished - 2016 Jan 1

    Fingerprint

    ornithine cyclodeaminase
    Arginase
    Proline
    Arginine
    Mixed Function Oxygenases
    Ornithine
    Hydroxyproline
    Enzymes
    Magnetic Resonance Spectroscopy
    High Pressure Liquid Chromatography
    Escherichia coli

    Keywords

    • (2S,3S)-3-Hydroxyornithine
    • Arginase
    • Arginine hydroxylase
    • Mesorhizobium loti
    • Ornithine cyclodeaminase
    • trans-3-Hydroxy-l-proline

    ASJC Scopus subject areas

    • Biotechnology
    • Applied Microbiology and Biotechnology

    Cite this

    Development of a multi-enzymatic cascade reaction for the synthesis of trans-3-hydroxy-l-proline from l-arginine. / Hara, Ryotaro; Kitatsuji, Saki; Yamagata, Kai; Kino, Kuniki.

    In: Applied Microbiology and Biotechnology, Vol. 100, No. 1, 01.01.2016, p. 243-253.

    Research output: Contribution to journalArticle

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    AB - Naturally occurring l-hydroxyproline in its four regio- and stereoisomeric forms has been explored as a possible precursor for pharmaceutical agents, yet the selective synthesis of trans-3-hydroxy-l-proline has not been achieved. Our aim was to develop a novel biocatalytic asymmetric method for the synthesis of trans-3-hydroxy-l-proline. So far, we focused on the rhizobial arginine catabolic pathway: arginase and ornithine cyclodeaminase are involved in l-arginine degradation to l-proline via l-ornithine. We hypothesized that trans-3-hydroxy-l-proline should be synthesized if arginase and ornithine cyclodeaminase act on (2S,3S)-3-hydroxyarginine and (2S,3S)-3-hydroxyornithine, respectively. To test this hypothesis, we cloned the genes of l-arginine 3-hydroxylase, arginase, and ornithine cyclodeaminase and overexpressed them in Escherichia coli, with subsequent enzyme purification. After characterization and optimization of each enzyme, a three-step procedure involving l-arginine 3-hydroxylase, arginase, and ornithine cyclodeaminase (in this order) was performed using l-arginine as a starting substrate. At the second step of the procedure, putative hydroxyornithine was formed quantitatively by arginase from (2S,3S)-3-hydroxyarginine. Nuclear magnetic resonance and chiral high-performance liquid chromatography analyses revealed that the absolute configuration of this compound was (2S,3S)-3-hydroxyornithine. In the last step of the procedure, trans-3-hydroxy-l-proline was synthesized selectively by ornithine cyclodeaminase from (2S,3S)-3-hydroxyornithine. Thus, we successfully developed a novel synthetic route, comprised of three reactions, to convert l-arginine to trans-3-hydroxy-l-proline. The excellent selectivity makes this procedure simpler and more efficient than conventional chemical synthesis.

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