Using mutability landscapes of a promiscuous tautomerase to guide the engineering of enantioselective Michaelases

Jan-Ytzen van der Meer, Harshwardhan Poddar, Bert-Jan Baas, Yufeng Miao, Mehran Rahimi, Andreas Kunzendorf, Ronald van Merkerk, Pieter G Tepper, Edzard M Geertsema, Andy-Mark W H Thunnissen, Wim J Quax, Gerrit J Poelarends

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Abstract

The Michael-type addition reaction is widely used in organic synthesis for carbon-carbon bond formation. However, biocatalytic methodologies for this type of reaction are scarce, which is related to the fact that enzymes naturally catalysing carbon-carbon bond-forming Michael-type additions are rare. A promising template to develop new biocatalysts for carbon-carbon bond formation is the enzyme 4-oxalocrotonate tautomerase, which exhibits promiscuous Michael-type addition activity. Here we present mutability landscapes for the expression, tautomerase and Michael-type addition activities, and enantioselectivity of 4-oxalocrotonate tautomerase. These maps of neutral, beneficial and detrimental amino acids for each residue position and enzyme property provide detailed insight into sequence-function relationships. This offers exciting opportunities for enzyme engineering, which is illustrated by the redesign of 4-oxalocrotonate tautomerase into two enantiocomplementary 'Michaelases'. These 'Michaelases' catalyse the asymmetric addition of acetaldehyde to various nitroolefins, providing access to both enantiomers of γ-nitroaldehydes, which are important precursors for pharmaceutically active γ-aminobutyric acid derivatives.

Original languageEnglish
Article number10911
Number of pages16
JournalNature Communications
Volume7
DOIs
Publication statusPublished - 2016

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