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

Rahimi, M., van der Meer, J-Y., Geertsema, E. M. & Poelarends, G. J. Jul-2017 In : ChemBioChem. p. 1435-1441

Research output: Scientific - peer-reviewArticle

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.

Original languageEnglish
Pages (from-to)1435-1441
JournalChemBioChem
Early online date20-Apr-2017
StatePublished - Jul-2017

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  • Journal Article

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