Tunable, post-translational hydroxylation of collagen Domains in Escherichia coli

ACS Chem Biol. 2011 Apr 15;6(4):320-4. doi: 10.1021/cb100298r. Epub 2011 Jan 14.


Prolyl 4-hydroxylases are ascorbate-dependent oxygenases that play key roles in a variety of eukaryotic biological processes including oxygen sensing, siRNA regulation, and collagen folding. They perform their functions by catalyzing the post-translational hydroxylation of specific proline residues on target proteins to form (2S,4R)-4-hydroxyproline. Thus far, the study of these post-translational modifications has been limited by the lack of a prokaryotic recombinant expression system for producing hydroxylated proteins. By introducing a biosynthetic shunt to produce ascorbate-like molecules in Eschericia coli cells that heterologously express human prolyl 4-hydroxylase (P4H), we have created a strain of E. coli that produces collagenous proteins with high levels of (2S,4R)-4-hydroxyproline. Using this new system, we have observed hydroxylation patterns indicative of a processive catalytic mode for P4H that is active even in the absence of ascorbate. Our results provide insights into P4H enzymology and create a foundation for better understanding how post-translational hydroxylation affects proteins.

Publication types

  • Letter
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ascorbic Acid / genetics
  • Ascorbic Acid / metabolism*
  • Chromatography, Liquid
  • Collagen / chemistry
  • Collagen / genetics
  • Collagen / metabolism*
  • Escherichia coli* / genetics
  • Escherichia coli* / metabolism
  • Gene Expression
  • Genetic Engineering / methods*
  • Humans
  • Hydroxylation
  • Hydroxyproline / metabolism
  • Mass Spectrometry
  • Plasmids / genetics
  • Plasmids / metabolism
  • Procollagen-Proline Dioxygenase / analysis
  • Procollagen-Proline Dioxygenase / chemistry
  • Procollagen-Proline Dioxygenase / genetics
  • Procollagen-Proline Dioxygenase / metabolism*
  • Proline / metabolism*
  • Protein Processing, Post-Translational
  • Transformation, Bacterial


  • Collagen
  • Proline
  • Procollagen-Proline Dioxygenase
  • Ascorbic Acid
  • Hydroxyproline