Int. J. Mol. Sci. 2015, 16(11), 26953-26963; doi:10.3390/ijms161126007
Alteration of the Donor/Acceptor Spectrum of the (S)-Amine Transaminase from Vibrio fluvialis
1
Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, 17487 Greifswald, Germany
2
Bio-Prodict, Nieuwe Marktstraat 54E, 6511 AA Nijmegen, The Netherlands
3
Protein Biochemistry, Institute of Biochemistry, University of Greifswald, 17487 Greifswald, Germany
*
Author to whom correspondence should be addressed.
Academic Editor: Vladimír Křen
Received: 9 October 2015 / Revised: 27 October 2015 / Accepted: 5 November 2015 / Published: 11 November 2015
(This article belongs to the Special Issue Molecular Biocatalysis)
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. View Full-TextKeywords:
amine transaminase; Vibrio fluvialis; amine; protein design; library creation
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Genz, M.; Vickers, C.; van den Bergh, T.; Joosten, H.-J.; Dörr, M.; Höhne, M.; Bornscheuer, U.T. Alteration of the Donor/Acceptor Spectrum of the (S)-Amine Transaminase from Vibrio fluvialis. Int. J. Mol. Sci. 2015, 16, 26953-26963.
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