Engineering a Promiscuous Tautomerase into a More Efficient Aldolase for Self-Condensations of Linear Aliphatic Aldehydes

4 years ago

Engineering a Promiscuous Tautomerase into a More Efficient Aldolase for Self-Condensations of Linear Aliphatic Aldehydes

Jan-Ytzen van der Meer, Edzard M. Geertsema, Gerrit J. Poelarends, Mehran Rahimi

The enzyme 4-oxalocrotonate tautomerase (4-OT) from Pseudomonas putida mt-2 takes part in a catabolic pathway for aromatic hydrocarbons, where it catalyzes the conversion of 2hydroxyhexa-2,4-dienedioate into 2-oxohexa-3-enedioate. This tautomerase can also promiscuously catalyze carbon–carbon bond-forming reactions, including various types of aldol reactions, by using its amino-terminal proline as a key catalytic residue. Here, we used systematic mutagenesis to identify two hotspots in 4-OT (Met45 and Phe50) at which single mutations give marked improvements in aldolase activity for the self-condensation of propanal. Activity screening of a focused library in which these two hotspots were varied led to the discovery of a 4-OT variant (M45Y/F50V) with strongly enhanced aldolase activity in the self-condensation of linear aliphatic aldehydes, such as acetaldehyde, propanal, and butanal, to yield α,β-unsaturated aldehydes. With both propanal and benzaldehyde, this double mutant, unlike the previously constructed single mutant F50A, mainly catalyzes the self-condensation of propanal rather than the cross-condensation of propanal and benzaldehyde, thus indicating that it indeed has altered substrate specificity. This variant could serve as a template to create new biocatalysts that lack dehydration activity and possess further enhanced aldolase activity, thus enabling the efficient enzymatic self-coupling of aliphatic aldehydes. Engineering a promiscuous enzyme: We have used a systematic mutagenesis strategy, followed by combinatorial mutagenesis of identified “hotspot” positions and activity screening, to greatly enhance the aldolase activity of 4-oxalocrotonate tautomerase for the self-condensation of linear aliphatic aldehydes thus yielding α,β-unsaturated aldehydes.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/cbic.201700121