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

ACS Synth Biol. 2015 Apr 17;4(4):383-92. doi: 10.1021/sb500247a. Epub 2014 Sep 5.


Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Proline hydroxylases are representative members of the nonheme Fe(2+)/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.

Keywords: hydroxy-l-pipecolic acid; hydroxylation; hydroxyproline; proline hydroxylase; protein engineering.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins* / chemistry
  • Bacterial Proteins* / metabolism
  • Crystallography, X-Ray
  • Hydroxylation
  • Mesorhizobium / enzymology*
  • Pipecolic Acids* / chemistry
  • Pipecolic Acids* / metabolism
  • Prolyl Hydroxylases* / chemistry
  • Prolyl Hydroxylases* / metabolism
  • Protein Structure, Tertiary
  • Sinorhizobium meliloti / enzymology*


  • Bacterial Proteins
  • Pipecolic Acids
  • Prolyl Hydroxylases
  • pipecolic acid