Crystal structure and reaction mechanism of urate oxidase from Bacillus sp.

芽孢杆菌尿酸氧化酶的晶体结构和反应机制。

• 批准号: 12660099
• 负责人: HIBI Takao
• 金额: $ 1.98万
• 依托单位: Fukui Prefectural University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660099/
• 关键词: uricacid; thermostability; T-fold motif; intersubmit interraction; X-ray crystallography; Structural Biology; Oxidase; Uricase; Thermostability; 尿酸; X線結晶構造解析; 酸化還元酵素; ウリカーゼ; X線結晶解析; 酸素ラジカル

Crystal structure of urate oxidase from Bacillus Sp. (bUOX) has been determined at 2.2A resolution. X-Ray diffraction data for the crystals were collected using a Mar CCD camera and synchrotron radition on beam line BL41XU (Spring-8, Harima, Japan). The crystals belong to space group P2_12_12 with unitcell parameters a=133.5, b=144.5, c=78.8A.The structure of UOX-8-azaxanthine complex has been solved by molecular replacement techniques. The refined structure of UOX from Asperigillus flavus (PDS code 1UOX; Colloc'h et al. (1997)) was used as our initial model, all the residues of which were replaced with alanines. Restrained refinement with Refmac5 (CCP4 Project No.4 (1999)) resulted in an R-value of 22% for the data (PDB code: 1J2G). Main chain structures of the loops at tetrametric interface are significantly different from those of A. flavus UOX.Instability of the b UOX crystal quality in changing salt concentration prevents from preparing Xe derivatives. However, another salt effect to bUOX was found that its thermal stability increases with salt concentration. Interestingly, one of the loops at the tetrameric interface contains R298 and a sulfate ion seems to bridge the subunits related with a non-crystallographic two-fold sysmmetry.

来自芽孢杆菌的尿酸氧化酶的晶体结构。 （Buox）已在2.2a分辨率下确定。使用MAR CCD摄像头和Beam Line BL41XU（Spring-8，Harima，Japan）上收集晶体的X射线衍射数据。晶体属于空间群P2_12_12，带有unitCell参数a = 133.5，b = 144.5，c = 78.8a。通过分子替代技术解决了UOX-8-氮杂蛋白复合物的结构。 Uox的精致结构（PDS代码1uox; Colloc'h等人（1997））被用作我们的初始模型，所有残基都被丙氨酸代替。 REFMAC5（CCP4项目编号4（1999））的限制改进导致数据的R值为22％（PDB代码：1J2G）。四聚界面上环的主链结构与Flavus Uox的界面显着不同。盐浓度变化的B UOX晶体质量的稳定性阻止了制备XE衍生物。然而，发现Buox的另一种盐效应是，其热稳定性随盐浓度而增加。有趣的是，四聚体界面上的一个环包含R298，硫酸盐离子似乎桥接了与非晶体学相关的亚基，这是两倍的Sysmmetry。

项目成果

T. Hibi: "Crystal stracture analysis of urate oxidase from Bacrllus sp."Spring 8 User Experimental heport. 8. 208 (2002)

T. Hibi：“芽孢杆菌属尿酸氧化酶的晶体结构分析”Spring 8 用户实验报告。

Y. Rishiya: "A pruification Method of the diagnostic enryne Bacillus Chicase using magnetic beads and non-specific protease"Protin Espression and purification. 25. 426-429 (2002)

Y. Rishiya：“使用磁珠和非特异性蛋白酶的诊断 enryne Bacillus Chicase 的纯化方法”蛋白质表达和纯化。

T. Hibi: "Enzymatic assay of Histamine by Amperometine Detectrin if H_2O_2 with knowdase-based Semsor"Biosci, Biothechrol. Biochem. 64. 1963-1966 (2002)

T. Hibi：“使用基于已知酶的 Semsor 进行 H_2O_2 时，通过 Amperometine Detectrin 进行组胺酶测定”Biosci，Biothechrol。

Y.Nishiya: "The full DNA sequence of the gene encoding the diagnostic Bacillus uricase"J. Anal. Bio-Sci. 23. 443-446 (2000)

Y.Nishiya：“编码诊断性尿酸芽孢杆菌基因的完整 DNA 序列”J.

T.Hibi: "Enzymatic Assay of Histamine by Amperometric Detection of H_2O_2 with a Peroxidase-based Sensor"Biosci. Biotechnol. Biochem. 64. 1963-1966 (2002)

T.Hibi：“使用基于过氧化物酶的传感器通过安培检测 H_2O_2 进行组胺酶法测定”Biosci。