A Rational Active-Site Redesign Converts a Decarboxylase into a C=C Hydratase: “Tethered Acetate” Supports Enantioselective Hydration of 4-Hydroxystyrenes
A Rational Active-Site Redesign Converts a Decarboxylase into a C=C Hydratase: “Tethered Acetate” Supports Enantioselective Hydration of 4-Hydroxystyrenes
复制标题
合理的活性位点重新设计将脱羧酶转化为 C=C 水合酶：“束缚乙酸酯”支持 4-羟基苯乙烯的对映选择性水合
作者：
Stefan E. Payer;Hannah Pollak;S. M. Glueck;K. Faber
肠杆菌阿魏酸脱羧酶催化 4-羟基苯乙烯的混杂区域选择性和立体选择性水合。 (FDC_Es) 取决于活性位点中结合的碳酸氢盐，其充当激活水分子以对醌甲基化物亲电子试剂进行亲核攻击的质子中继。这种“辅助因子”对于提高 (S) 配置的苄醇产品的转化率和高立体选择性至关重要。使用简单的脂肪族羧酸作为添加剂观察到类似的效果。通过用来自相邻氨基酸的新引入的侧链羧酸盐替换碳酸氢盐或乙酸盐“辅因子”，对活性位点进行合理的重新设计，产生了有效充当 C=C 水合酶的突变体。缬氨酸 46 向谷氨酸或天冬氨酸的单点突变将水合活性提高了 40%，并且在没有碳酸氢盐或乙酸盐的情况下将立体选择性提高了 39 倍。
The promiscuous regio- and stereoselective hydration of 4-hydroxystyrenes catalyzed by ferulic acid decarboxylase from Enterobacter sp. (FDC_Es) depends on bicarbonate bound in the active site, which serves as a proton relay activating a water molecule for nucleophilic attack on a quinone methide electrophile. This “cofactor” is crucial for achieving improved conversions and high stereoselectivities for (S)-configured benzylic alcohol products. Similar effects were observed with simple aliphatic carboxylic acids as additives. A rational redesign of the active site by replacing the bicarbonate or acetate “cofactor” with a newly introduced side-chain carboxylate from an adjacent amino acid yielded mutants that efficiently acted as C=C hydratases. A single-point mutation of valine 46 to glutamate or aspartate improved the hydration activity by 40% and boosted the stereoselectivity 39-fold in the absence of bicarbonate or acetate.
扩展酶的宇宙：通过机制引导的定向进化来访问非自然反应。
影响因子：
16.6
作者：
Renata, Hans;Wang, Z. Jane;Arnold, Frances H.
通讯作者：
Arnold, Frances H.