S‐Selective Mixed Carboligation by Structure‐Based Design of the Pyruvate Decarboxylase from Acetobacter pasteurianus
S‐Selective Mixed Carboligation by Structure‐Based Design of the Pyruvate Decarboxylase from Acetobacter pasteurianus
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DOI：
10.1002/cctc.201100054
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发表时间：
2011-10
期刊：
影响因子：
4.5
通讯作者：
Dörte Rother (neé Gocke);G. Kolter;Tina Gerhards;C. L. Berthold;Ekaterina V. Gauchenova;Michael Knoll;J. Pleiss;Michael Müller;G. Schneider;M. Pohl
中科院分区：
文献类型：
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作者：
Dörte Rother (neé Gocke);G. Kolter;Tina Gerhards;C. L. Berthold;Ekaterina V. Gauchenova;Michael Knoll;J. Pleiss;Michael Müller;G. Schneider;M. Pohl
来自巴氏醋杆菌（Acetobacter pasteurianus）的硫胺素二磷酸（ThDP）依赖性丙酮酸脱羧酶（ApPDC）催化醛的碳化，在脂肪族供体和芳香族受体醛的混合碳化中以高化学选择性产生（R）-2-羟基酮。根据ApPDC的晶体结构和生化数据，我们绘制了活性位点。这使我们能够通过修饰残基E469和W388来设计具有定制催化活性的变体。虽然W388被较小的氨基酸交换产生了具有更高碳连接酶活性的变体，这是由于增加了对活性位点的访问，但E469交换为甘氨酸打开了ApPDC中所谓的芳香醛的S-口袋，从而改变了立体选择性。变体ApPDC-E469 G通过酶促碳化提供了获得（S）-苯乙酰基甲醇衍生物的途径，具有高达89%对映体过量的良好立体选择性。该变体很好地补充了ThDP依赖性酶的工具箱，该工具箱现在可以访问不对称芳香族交叉安息香样缩合的所有立体异构体和区域异构体。我们证明，这两个醛的活性位点的最佳稳定是必不可少的，以获得高产率和高选择性。
The thiamine diphosphate (ThDP)‐dependent pyruvate decarboxylase from Acetobacter pasteurianus (ApPDC) catalyzes the carboligation of aldehydes that yields (R)‐2‐hydroxy ketones with high chemoselectivity in mixed carboligations of aliphatic donor and aromatic acceptor aldehydes. On the basis of the crystal structure of ApPDC, which was determined to a resolution of 2.75 Å, and biochemical data, we mapped the active site. This enabled us to design variants with tailor‐made catalytic activities by modifications of the residues E469 and W388. Although the exchange of W388 by smaller amino acids yields variants with higher carboligase activity due to an increased access to the active site, the exchange of E469 to glycine opens the so‐called S‐pocket in ApPDC for aromatic aldehydes and thus alters the stereoselectivity. The variant ApPDC‐E469G provides access to (S)‐phenylacetylcarbinol derivatives by enzymatic carboligation with a good stereoselectivity of up to 89 % enantiomeric excess. The variant nicely complements the toolbox of ThDP‐dependent enzymes, which now gives access to all stereo‐ and regioisomers of the asymmetric aliphatic–aromatic cross‐benzoin‐like condensation. We prove that optimal stabilization of both aldehydes in the active site is essential to gain high yields and high selectivities.