STD-NMR-Based Protein Engineering of the Unique Arylpropionate-Racemase AMDase G74C
STD-NMR-Based Protein Engineering of the Unique Arylpropionate-Racemase AMDase G74C
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DOI：
10.1002/cbic.201500253
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发表时间：
2015-09-07
期刊：
影响因子：
3.2
通讯作者：
Kourist, Robert
中科院分区：
文献类型：
--
作者：
Gassmeyer, Sarah Katharina;Yoshikawa, Hiroyuki;Kourist, Robert
结构引导的蛋白质工程实现了独特的消旋酶AMDase G74C的变体，在几种芳香脂肪族羧酸的消旋活性提高了40倍。用饱和转移差核磁共振(STD-NMR)研究了催化过程中的底物结合情况。底物上的所有原子都与酶有相互作用。STD-核磁共振测量显示，在有和没有分子转化的实验中，有明显的核Overhaser效应。光谱分析鉴定了几个氨基酸残基，它们的取代提高了G74C的活性。单一氨基酸交换适度提高活性；结构导向饱和诱变产生四重突变体，反应速度提高40倍。这项研究将STD-核磁共振作为分析催化活性体系中酶-底物相互作用的通用工具，并用于指导蛋白质工程。
Structure-guided protein engineering achieved a variant of the unique racemase AMDase G74C, with 40-fold increased activity in the racemisation of several arylaliphatic carboxylic acids. Substrate binding during catalysis was investigated by saturation-transfer-difference NMR (STD-NMR) spectroscopy. All atoms of the substrate showed interactions with the enzyme. STD-NMR measurements revealed distinct nuclear Overhauser effects in experiments with and without molecular conversion. The spectroscopic analysis led to the identification of several amino acid residues whose substitutions increased the activity of G74C. Single amino acid exchanges increased the activity moderately; structure-guided saturation mutagenesis yielded a quadruple mutant with a 40 times higher reaction rate. This study presents STD-NMR as versatile tool for the analysis of enzyme-substrate interactions in catalytically competent systems and for the guidance of protein engineering.