Characterization of novel 2-oxoglutarate dependent dioxygenases converting L-proline to cis-4-hydroxy-l-proline

Biochem Biophys Res Commun. 2009 Feb 20;379(4):882-6. doi: 10.1016/j.bbrc.2008.12.158. Epub 2009 Jan 6.


Hydroxyprolines are valuable chiral building blocks for organic synthesis of pharmaceuticals. Several microorganisms producing L-proline trans-4- and cis-3-hydroxylase were discovered and these enzymes were applied to the industrial production of trans-4- and cis-3-hydroxy-L-proline, respectively. Meanwhile, other hydroxyproline isomers, cis-4- and trans-3-hydroxy-L-proline, were not easily available because the corresponding hydroxylase have not been discovered. Herein we report novel L-proline cis-4-hydroxylases converting free L-proline to cis-4-hydroxy-L-proline. Two genes encoding uncharacterized proteins from Mesorhizobium loti and Sinorhizobium meliloti were cloned and overexpressed in Escherichia coli, respectively. The functions of purified proteins were investigated in detail, and consequently we detected L-proline cis-4-hydroxylase activity in both proteins. Likewise L-proline trans-4-, cis-3-hydroxylase and prolyl hydroxylase, these enzymes belonged to a 2-oxoglutarate dependent dioxygenase family and required a non-heme ferrous ion. Although their reaction mechanisms were similar to other hydroxylases, the amino acid sequence homology was not observed (less than 40%).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alphaproteobacteria / enzymology*
  • Alphaproteobacteria / genetics
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Hydrogen-Ion Concentration
  • Hydroxyproline / biosynthesis*
  • Ketoglutarate Dehydrogenase Complex / chemistry*
  • Ketoglutarate Dehydrogenase Complex / genetics
  • Ketoglutarate Dehydrogenase Complex / isolation & purification
  • Proline / metabolism*
  • Sinorhizobium meliloti / enzymology
  • Sinorhizobium meliloti / genetics
  • Temperature


  • Proline
  • Ketoglutarate Dehydrogenase Complex
  • Hydroxyproline