Engineering a Promiscuous Tautomerase into a More Efficient Aldolase for Self‐Condensations of Linear Aliphatic Aldehydes
Abstract: 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.
- Location
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Deutsche Nationalbibliothek Frankfurt am Main
- Extent
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Online-Ressource
- Language
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Englisch
- Bibliographic citation
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Engineering a Promiscuous Tautomerase into a More Efficient Aldolase for Self‐Condensations of Linear Aliphatic Aldehydes ; volume:18 ; number:14 ; year:2017 ; pages:1435-1441 ; extent:7
ChemBioChem ; 18, Heft 14 (2017), 1435-1441 (gesamt 7)
- Creator
- DOI
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10.1002/cbic.201700121
- URN
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urn:nbn:de:101:1-2022092308434199333837
- Rights
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Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
- Last update
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15.08.2025, 7:26 AM CEST
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Deutsche Nationalbibliothek. If you have any questions about the object, please contact the data provider.
Associated
- Rahimi, Mehran
- van der Meer, Jan‐Ytzen
- Geertsema, Edzard M.
- Poelarends, Gerrit
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