Genz Maika, Vickers Clare, van den Bergh Tom, Joosten Henk-Jan, Dörr Mark, Höhne Matthias, Bornscheuer Uwe T
Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, 17487 Greifswald, Germany.
Bio-Prodict, Nieuwe Marktstraat 54E, 6511 AA Nijmegen, The Netherlands.
Int J Mol Sci. 2015 Nov 11;16(11):26953-63. doi: 10.3390/ijms161126007.
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.
为了改变（S）-选择性胺转氨酶（ATA）的胺供体/受体谱，构建了一个基于河流弧菌ATA的文库，该文库针对靠近活性位点的四个残基（L56、W57、R415和L417）。一个由3DM衍生的包含I类磷酸吡哆醛（PLP）依赖性酶的比对，使得能够确定假定决定底物特异性的位置。这些位置用疏水性氨基酸的特定字母表进行定点诱变，以将胺：α-酮酸转移酶转化为胺：醛转移酶。对1200个变体的筛选揭示了三个命中变体，它们显示出胺供体/受体谱向脂肪醛（主要是戊醛）的偏移，以及pH谱的改变。有趣的是，所有三个命中变体尽管是独立发现的，但都包含相同的R415L突变以及额外的W57F和L417V取代。
