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

Nat Commun. 2016 Mar 8:7:10911. doi: 10.1038/ncomms10911.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biocatalysis
  • Crotonates / chemistry*
  • Crotonates / metabolism
  • Isomerases / chemistry*
  • Isomerases / genetics
  • Isomerases / metabolism
  • Kinetics
  • Protein Engineering*
  • Stereoisomerism
  • Substrate Specificity

Substances

  • Crotonates
  • oxalocrotonate
  • 4-oxalocrotonate tautomerase
  • Isomerases