title: Alteration of the Donor/Acceptor Spectrum of the (S)-Amine Transaminase from Vibrio fluvialis.
abstract: To alter the amine donor/acceptor spectrum of an (S)-selective amine transaminase (ATA), a library based on the Vibrio fluvialis ATA targeting four residues close to the active site (L56, W57, R415 and L417) was created. A 3DM-derived alignment comprising fold class I pyridoxal-5'-phosphate (PLP)-dependent enzymes allowed identification of positions, which were assumed to determine substrate specificity. These positions were targeted for mutagenesis with a focused alphabet of hydrophobic amino acids to convert an amine:α-keto acid transferase into an amine:aldehyde transferase. Screening of 1200 variants revealed three hits, which showed a shifted amine donor/acceptor spectrum towards aliphatic aldehydes (mainly pentanal), as well as an altered pH profile. Interestingly, all three hits, although found independently, contained the same mutation R415L and additional W57F and L417V substitutions.
authors.affiliation: Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, 17487 Greifswald, Germany. maika.genz@uni-greifswald.de.
authors.forename: Maika
authors.initials: M
authors.lastname: Genz
authors.affiliation: Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, 17487 Greifswald, Germany. clare.vickers@uni-greifswald.de.
authors.forename: Clare
authors.initials: C
authors.lastname: Vickers
authors.affiliation: Bio-Prodict, Nieuwe Marktstraat 54E, 6511 AA Nijmegen, The Netherlands. tvandenbergh@bio-prodict.nl.
authors.forename: Tom
authors.initials: T
authors.lastname: van den Bergh
authors.affiliation: Bio-Prodict, Nieuwe Marktstraat 54E, 6511 AA Nijmegen, The Netherlands. joosten@bio-prodict.nl.
authors.forename: Henk-Jan
authors.initials: HJ
authors.lastname: Joosten
authors.affiliation: Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, 17487 Greifswald, Germany. mark.doerr@uni-greifswald.de.
authors.forename: Mark
authors.initials: M
authors.lastname: Dörr
authors.affiliation: Protein Biochemistry, Institute of Biochemistry, University of Greifswald, 17487 Greifswald, Germany. matthias.hoehne@uni-greifswald.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Höhne
authors.affiliation: Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, 17487 Greifswald, Germany. uwe.bornscheuer@uni-greifswald.de.
authors.forename: Uwe T
authors.initials: UT
authors.lastname: Bornscheuer
mesh_terms: D000588:Amines / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007651:Keto Acids / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D011485:Protein Binding; D013379:Substrate Specificity; D000637:Transaminases / Q000737:chemistry* / Q000378:metabolism*; D014733:Vibrio / Q000201:enzymology / Q000378:metabolism*
chemical_list: D000588:Amines; D007651:Keto Acids; D000637:Transaminases
keywords: Vibrio fluvialis; amine; amine transaminase; library creation; protein design