Selective oxidative demethylation of veratric acid to vanillic acid by CYP199A4 from Rhodopseudomonas palustris HaA2

Mol Biosyst. 2010 Jan;6(1):206-14. doi: 10.1039/b913487e. Epub 2009 Sep 14.


CYP199A4 (RPB3613) from Rhodopseudomonas palustris HaA2 is a heme monooxygenase that catalyzes the hydroxylation of para-substituted benzoic acids. Monooxygenase activity of CYP199A4 can be reconstituted in a Class I electron transfer chain with an associated [2Fe-2S] ferredoxin, HaPux, (RPB3614) and the flavin-dependent reductase, HaPuR, (RPB3656) that is not associated with a CYP gene. CYP199A4 and the ferredoxin HaPux are produced in greater quantities using recombinant Escherichia coli expression systems when compared to the equivalent proteins in the closely related CYP199A2-Pux-PuR Class I system from R. palustris CGA009. HaPuR and HaPux can also replace PuR and Pux in supporting the CYP199A2 enzyme turnover with high activity. Whole-cell in vivo substrate oxidation systems for CYP199A4 and CYP199A2 with HaPux and HaPuR as the electron transfer proteins have been constructed. These E. coli systems were capable of selectively demethylating veratric acid at the para position to produce vanillic acid at rates of up to 15.3 microM (g-cdw)(-1) min(-1) and yields of up to 1.2 g L(-1).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Ferredoxins / metabolism
  • Kinetics
  • Methylation
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / metabolism*
  • Rhodopseudomonas / genetics
  • Rhodopseudomonas / metabolism*
  • Vanillic Acid / analogs & derivatives*
  • Vanillic Acid / metabolism*


  • 2Fe-2S ferredoxin
  • Bacterial Proteins
  • Ferredoxins
  • veratric acid
  • Mixed Function Oxygenases
  • Vanillic Acid