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

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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.

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Acknowledgements

This work was supported in part by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 15 K18677 (to R.H.).

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The authors declare that they have no competing interests.

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Authors and Affiliations

  1. Research Institute for Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo, 169-8555, Japan

    Ryotaro Hara & Kuniki Kino

  2. Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo, 169-8555, Japan

    Saki Kitatsuji, Kai Yamagata & Kuniki Kino

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  1. Ryotaro Hara
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  2. Saki Kitatsuji
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  3. Kai Yamagata
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Correspondence to Ryotaro Hara.

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Hara, R., Kitatsuji, S., Yamagata, K. et al. Development of a multi-enzymatic cascade reaction for the synthesis of trans-3-hydroxy-l-proline from l-arginine. Appl Microbiol Biotechnol 100, 243–253 (2016). https://doi.org/10.1007/s00253-015-6992-4

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