National Center for
4X1C: Crystal Structure Of 4-ot From Pseudomonas Putida Mt-2 With An Enamine Adduct On The N-terminal Proline At 1.7 Angstrom Resolution
Evidence for the Formation of an Enamine Species during Aldol and Michael-type Addition Reactions Promiscuously Catalyzed by 4-Oxalocrotonate Tautomerase
Chembiochem (2015) 16 p.738-741
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 gamma-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.