Crystallization and preliminary X-ray diffraction analysis of domain chimeric L-(2S, 3S)-butanediol dehydrogenase
Crystallization and preliminary X-ray diffraction analysis of domain chimeric L-(2S, 3S)-butanediol dehydrogenase
复制标题
域嵌合L-(2S,3S)-丁二醇脱氢酶的结晶和初步X射线衍射分析
DOI：
10.1107/s2053230x13032755
复制
发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Ui S
中科院分区：
文献类型：
--
作者：
Shimegi T;Oyama T;Ohtsuki T;Kurisu G;Kusunoki M;Ui S
通过交换这两种BDH的相应结构域，构建了结构域嵌合的1 - 2，3-丁二醇脱氢酶（嵌合体1-BDH），其被设计为同时具有1-BDH的S-构型特异性和meso-BDH的稳定性。然而，嵌合体I-BDH具有比基于两种原始酶的预期更低的酶功能。为了阐明稳定性和底物特异性降低的原因，进行了蛋白质的结晶。通过硫酸铵分级分离和三个柱色谱步骤将嵌合体I-BDH纯化至均匀，并使用悬滴气相扩散法结晶。晶体属于空间群C2221，衍射同步辐射到1.58 μ m的分辨率，最有可能包含两个分子的不对称单位。
A domain-chimeric l-2,3-butanediol dehydrogenase (chimera l-BDH), which was designed to possess both the S-configuration specificity of l-BDH and the stability of meso-BDH, was constructed by exchanging the respective domains of these two BDHs. However, chimera l-BDH possessed a lower enzymatic function than expected based on the two original enzymes. To elucidate the causes of the decreased stability and substrate specificity, crystallization of the protein was performed. Chimera l-BDH was purified to homogeneity via ammonium sulfate fractionation and three column-chromatography steps, and was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group C2221, diffracted synchrotron radiation to 1.58 Å resolution and were most likely to contain two molecules in the asymmetric unit.