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

*この研究の対応する著者

研究成果: Article査読

22 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)383-392
ページ数10
ジャーナルACS Synthetic Biology
4
4
DOI
出版ステータスPublished - 2015 4 17

ASJC Scopus subject areas

  • 生体医工学
  • 生化学、遺伝学、分子生物学(その他)

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