Engineering a thermostable transketolase for unnatural conversion of (2S)-hydroxyaldehydes
Engineering a thermostable transketolase for unnatural conversion of (2S)-hydroxyaldehydes
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DOI：
10.1002/adsc.201500207
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发表时间：
2015-05
影响因子：
5.4
通讯作者：
Juliane Abdoul Zabar;Marion Lorillière;D. Yi;T. Saravanan;T. Devamani;L. Nauton;F. Charmantray
中科院分区：
文献类型：
--
作者：
Juliane Abdoul Zabar;Marion Lorillière;D. Yi;T. Saravanan;T. Devamani;L. Nauton;F. Charmantray
来自不同来源（包括大肠杆菌和酵母）的转酮醇酶（TK）已被描述为对（2 R）-羟基醛底物具有完全对映体特异性。发现嗜热脂肪土芽孢杆菌（TKgst）的热稳定TK对（2S）-羟基化醛表现出轻微反应性。为了通过定向蛋白质进化来提高这种活性，我们通过在两个关键位置L382和D470上的位点饱和诱变来构建TKgst变体的文库。最好的TKgst双突变体L382 D/D470 S分别对作为受体底物的L-乳醛和L-甘油醛显示高达4倍和5倍的活性。通过一步合成有价值的L-核酮糖及其具有L-β-（3S，4S）构型的5-脱氧类似物证明了该突变体的重复效用，这些是以前使用普通TK来源无法获得的。
Transketolase (TK) from various origins (including Escherichia coli and yeast) has been described to be fully enantiomer specific for (2R)-hydroxyaldehyde substrates. A thermostable TK from Geobacillus stearothermophilus (TKgst) was found to display a minor reactivity for (2S)-hydroxylated aldehydes. To improve this activity by directed protein evolution, we have built a library of TKgst variants by site saturation mutagenesis on two key positions L382 and D470. The best TKgst double mutant L382D/D470S shows up to 4- and 5-fold higher activities towards L-lactaldehyde and L-glyceraldehyde as acceptor substrates, respectively. Preparative utility of this mutant was demonstrated by the one-step synthesis of valuable L-ribulose and its 5-deoxy analogue with the L-erythro (3S,4S) configuration, which were previously inaccessible by using common TK sources.