Evidence for the formation of an enamine species during aldol and Michael-type addition reactions promiscuously catalyzed by 4-oxalocrotonate tautomerase

Chembiochem. 2015 Mar 23;16(5):738-41. doi: 10.1002/cbic.201402687. Epub 2015 Feb 26.


The enzyme 4-oxalocrotonate tautomerase (4-OT), which has a catalytic N-terminal proline residue (Pro1), can promiscuously catalyze various carbon-carbon bond-forming reactions, including aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde, and Michael-type addition of acetaldehyde to a wide variety of nitroalkenes to yield valuable γ-nitroaldehydes. To gain insight into how 4-OT catalyzes these unnatural reactions, we carried out exchange studies in D2 O, and X-ray crystallography studies. The former established that H-D exchange within acetaldehyde is catalyzed by 4-OT and that the Pro1 residue is crucial for this activity. The latter showed that Pro1 of 4-OT had reacted with acetaldehyde to give an enamine species. These results provide evidence of the mechanism of the 4-OT-catalyzed aldol and Michael-type addition reactions in which acetaldehyde is activated for nucleophilic addition by Pro1-dependent formation of an enamine intermediate.

Keywords: 4-oxalocrotonate tautomerase; H-D exchange; Michael addition; aldol reaction; enamine formation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldehydes / chemistry
  • Aldehydes / metabolism*
  • Amines / chemistry
  • Amines / metabolism*
  • Biocatalysis
  • Crystallography, X-Ray
  • Isomerases / chemistry
  • Isomerases / metabolism*
  • Models, Molecular
  • Molecular Structure


  • Aldehydes
  • Amines
  • 4-oxalocrotonate tautomerase
  • Isomerases