title: Using mutability landscapes of a promiscuous tautomerase to guide the engineering of enantioselective Michaelases.
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.
authors.affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands.
authors.forename: Jan-Ytzen
authors.initials: JY
authors.lastname: van der Meer
authors.affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands.
authors.forename: Harshwardhan
authors.initials: H
authors.lastname: Poddar
authors.affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands.
authors.forename: Bert-Jan
authors.initials: BJ
authors.lastname: Baas
authors.affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands.
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Miao
authors.affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands.
authors.forename: Mehran
authors.initials: M
authors.lastname: Rahimi
authors.affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kunzendorf
authors.affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands.
authors.forename: Ronald
authors.initials: R
authors.lastname: van Merkerk
authors.affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands.
authors.forename: Pieter G
authors.initials: PG
authors.lastname: Tepper
authors.affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands.
authors.forename: Edzard M
authors.initials: EM
authors.lastname: Geertsema
authors.affiliation: Department of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, Groningen 9747 AG, The Netherlands.
authors.forename: Andy-Mark W H
authors.initials: AM
authors.lastname: Thunnissen
authors.affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands.
authors.forename: Wim J
authors.initials: WJ
authors.lastname: Quax
authors.affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands.
authors.forename: Gerrit J
authors.initials: GJ
authors.lastname: Poelarends
mesh_terms: D055162:Biocatalysis; D003437:Crotonates / Q000737:chemistry* / Q000378:metabolism; D007535:Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D015202:Protein Engineering; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D003437:Crotonates; C002788:oxalocrotonate; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases