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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

21 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 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.

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

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

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