title: Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4-Oxalocrotonate Tautomerase More Effectively than Distant Mutations.
abstract: The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000-fold improvement in catalytic efficiency (kcat /Km ) and a >10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site.
authors.affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, 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, 9713, AV, Groningen, 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, 9713, AV, Groningen, The Netherlands.|Institute for Life Science & Technology, Hanze University of Applied Sciences, Zernikeplein 11, 9747, AS, Groningen, The Netherlands.
authors.forename: Edzard M
authors.initials: EM
authors.lastname: Geertsema
authors.affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, 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, 9713, AV, Groningen, 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, 9713, AV, Groningen, The Netherlands. g.j.poelarends@rug.nl.
authors.forename: Gerrit J
authors.identifier: 0000-0002-6917-6368
authors.initials: GJ
authors.lastname: Poelarends
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D003062:Codon; D007535:Isomerases / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D015202:Protein Engineering; D016958:Pseudomonas putida / Q000201:enzymology
chemical_list: D003062:Codon; D000446:Aldehyde-Lyases; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases
keywords: aldolase activity; catalytic promiscuity; mutagenesis; oxalocrotonate tautomerase; protein engineering