来自木兰假丝酵母的羰基还原酶S1的表达、纯化、结晶及X射线初步分析。
Expression, purification, crystallization and preliminary X-ray analysis of carbonyl reductase S1 from Candida magnoliae.
作者信息
Suwa Yoichi, Ohtsuka Jun, Miyakawa Takuya, Imai Fabiana Lica, Okai Masahiko, Sawano Yoriko, Yasohara Yoshihiko, Kataoka Michihiko, Shimizu Sakayu, Tanokura Masaru
机构信息
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
出版信息
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012 May 1;68(Pt 5):540-2. doi: 10.1107/S1744309112011645. Epub 2012 Apr 20.
The NADPH-dependent carbonyl reductase S1 from Candida magnoliae stereoselectively catalyzes the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE), which is a chiral compound valuable as a building block for pharmaceuticals. Carbonyl reductase S1 was expressed in Escherichia coli and purified by Ni-affinity, ion-exchange and size-exclusion chromatography. Crystals of carbonyl reductase S1 were obtained by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. X-ray diffraction data were collected to 1.90 Å resolution using a synchrotron-radiation source. The crystals belonged to space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 77.7, c = 307.5 Å. The asymmetric unit contained two molecules of the protein, with a solvent content of 44.2%.
来自大花假丝酵母的依赖烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的羰基还原酶S1可立体选择性地催化将4-氯-3-氧代丁酸乙酯（COBE）还原为（S）-4-氯-3-羟基丁酸乙酯（CHBE），CHBE是一种作为药物合成砌块很有价值的手性化合物。羰基还原酶S1在大肠杆菌中表达，并通过镍亲和、离子交换和尺寸排阻色谱法进行纯化。使用聚乙二醇400（PEG 400）作为沉淀剂，通过坐滴气相扩散法获得了羰基还原酶S1的晶体。使用同步辐射光源收集了分辨率为1.90 Å的X射线衍射数据。晶体属于空间群P6(1)22或P6(5)22，晶胞参数a = b = 77.7，c = 307.5 Å。不对称单元包含两个蛋白质分子，溶剂含量为44.2%。
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