A unique dye-decolorizing peroxidase, DyP, from Thanatephorus cucumeris Dec 1: heterologous expression, crystallization and preliminary X-ray analysis.
A unique dye-decolorizing peroxidase, DyP, from Thanatephorus cucumeris Dec 1: heterologous expression, crystallization and preliminary X-ray analysis.
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DOI：
10.1107/s0907444903025472
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发表时间：
2004
期刊：
影响因子：
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通讯作者：
Takao Sato;Shusaku Hara;T. Matsui;G. Sazaki;S. Saijo;Tadashi Ganbe;N. Tanaka;Y. Sugano;M. Shoda
中科院分区：
文献类型：
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作者：
Takao Sato;Shusaku Hara;T. Matsui;G. Sazaki;S. Saijo;Tadashi Ganbe;N. Tanaka;Y. Sugano;M. Shoda
染料脱色过氧化物酶 DyP 是脱色真菌 Thanatephorus cucumeris Dec 1 中的关键酶，可降解偶氮和蒽醌染料。克隆来自黄瓜T. cucumeris Dec 1的dyp基因，该基因与其他过氧化物酶基因同源性较低，并转化到米曲霉中，糖基化DyP得到高水平表达。使用 GST Endo F1 对纯化的 DyP 进行去糖基化，然后使用硫酸铵作为沉淀剂在 283 K 的强磁场 (10 T) 中结晶。从光子工厂（日本筑波）的天然晶体收集的分辨率为 2.96 A 的 X 射线衍射数据表明，该晶体属于六方空间群 P6(5)22，晶胞参数 a = b = 136.15，c = 363.46 A。晶体的不对称单元包含四个 DyP 分子，相应的马修斯系数 (V(M)) 为 2.50 A(3) Da(-1)且溶剂含量为51%。获得了 DyP 的重原子衍生物并计算了电子密度图。血红素是可见的，血红素和蛋白质之间的连续电子密度清楚地表明了近端组氨酸配体的位置。
The dye-decolorizing peroxidase DyP is a key enzyme in the decolorizing fungus Thanatephorus cucumeris Dec 1 that degrades azo and antraquinone dyes. The gene dyp from T. cucumeris Dec 1, which has low homology to other peroxidase genes, was cloned and transformed into Aspergillus oryzae and glycosylated DyP was expressed at high levels. Purified DyP was deglycosylated using GST Endo F1 and then crystallized in a strong magnetic field (10 T) at 283 K using ammonium sulfate as precipitant. X-ray diffraction data to 2.96 A resolution collected from a native crystal at the Photon Factory (Tsukuba, Japan) showed that the crystal belonged to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 136.15, c = 363.46 A. The asymmetric unit of the crystal contained four DyP molecules, with a corresponding Matthews coefficient (V(M)) of 2.50 A(3) Da(-1) and a solvent content of 51%. Heavy-atom derivatives of DyP have been obtained and electron-density maps have been calculated. The haem is visible and continuous electron density between the haem and protein clearly indicates the location of the proximal histidine ligand.