Catalytic promiscuity of a proline-based tautomerase: Aldolase activities and enzyme redesign

Numerous enzymes have been found to catalyze additional and completely different types of reactions relative to the natural activity they evolved for. This interesting phenomenon, called catalytic promiscuity, has proven to be a fruitful guide for the development of novel biocatalysts for organic synthesis purposes.
A remarkable example of an enzyme with catalytic promiscuity is 4-oxalocrotonate tautomerase (4-OT). This enzyme naturally catalyzes an enol-keto tautomerization step as part of a catabolic pathway for aromatic hydrocarbons, but can also promiscuously catalyze carbon-carbon bond-forming aldol and Michael-type addition reactions.
Mehran Rahimi investigated the mechanism by which 4-OT catalyzes these unnatural reactions. Hydrogen-deuterium exchange and crystallographic studies of the enzyme in the presence of acetaldehyde provided strong evidence that the characteristic active site proline residue (Pro-1) of 4-OT reacts with acetaldehyde to give a covalent enamine species. A reaction between this nucleophilic intermediate and an electrophilic substrate, such as benzaldehyde or trans-β-nitrostyrene, results in carbon-carbon bond formation.
Mehran Rahimi also discovered that 4-OT can promiscuously catalyze various inter- and intramolecular aldol reactions. By using a highly informative systematic mutagenesis strategy, ‘hotspot’ positions in 4-OT at which single mutations give a marked improvement in aldolase activity were identified. Activity screening of focused libraries in which only a few ‘hotspot’ positions were varied led to the discovery of 4-OT variants with strongly enhanced aldolase activities.
Taken together, the results presented in Mehran Rahimi’s thesis demonstrate that a catalytically promiscuous tautomerase is a good starting point to develop novel biocatalysts for synthetically useful aldol reactions.