Structurally informed site-directed mutagenesis of a stereochemically promiscuous aldolase to afford stereochemically complementary biocatalysts.
Structurally informed site-directed mutagenesis of a stereochemically promiscuous aldolase to afford stereochemically complementary biocatalysts.
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DOI：
10.1021/ja104412a
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发表时间：
2010-08
影响因子：
15
通讯作者：
S. Royer;Luke J. Haslett;S. Crennell;D. Hough;M. Danson;S. Bull
中科院分区：
文献类型：
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作者：
S. Royer;Luke J. Haslett;S. Crennell;D. Hough;M. Danson;S. Bull
2-酮-3-脱氧葡萄糖酸醛缩酶是一种高耐热性的I型醛缩酶，可以利用非磷酸化底物催化碳-碳键形成。然而，它在许多醛醇反应中表现出较差的非对构控制，包括其天然底物丙酮酸和d -甘油醛的反应，它们提供55:45的d -2-酮-3-脱氧葡萄糖酸盐（D-KDGlu）和d -2-酮-3-脱氧半乳糖酸盐（D-KDGal）混合物。我们利用与这些非对映异构体醛醇产物结合的醛缩酶的详细x射线晶体结构信息，选择性地靶向特定氨基酸进行突变，以快速产生立体化学互补突变体，催化(Re)-或(Si)-面选择性醛缩反应，从而提供具有良好非对映控制水平的D-KDGlu或D-KDGal。
2-Keto-3-deoxygluconate aldolase from the hyperthermophile Sulfolobus solfataricus is a highly thermostable type I aldolase that can catalyze carbon-carbon bond formation using nonphosphorylated substrates. However, it exhibits poor diastereocontrol in many of its aldol reactions, including the reaction of its natural substrates, pyruvate and D-glyceraldehyde, which afford a 55:45 mixture of D-2-keto-3-deoxygluconate (D-KDGlu) and D-2-keto-3-deoxy-galactonate (D-KDGal). We have employed detailed X-ray crystallographic structural information of this aldolase bound to these diastereoisomeric aldol products to selectively target specific amino acids for mutation for the rapid creation of stereochemically complementary mutants that catalyze either (Re)- or (Si)-facial selective aldol reactions to afford either D-KDGlu or D-KDGal with good levels of diastereocontrol.