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.

本文测定了芽孢杆菌尿酸氧化酶（bUOX）的晶体结构，分辨率为2.2A。使用Mar CCD照相机和同步加速器辐射在光束线BL 41 XU（Spring-8，Harima，Japan）上收集晶体的X射线衍射数据。晶体属P2_12_12空间群，晶胞参数a=133.5，B=144.5，c= 78.8 A。用分子置换技术解决了UOX-8-氮杂黄嘌呤配合物的结构。来自黄曲霉的UOX的精细结构（PDS代码1UOX; Colloc等人（1997））被用作我们的初始模型，其所有残基都被丙氨酸取代。使用Refmac 5（CCP 4 Project No.4（1999））进行的限制性细化导致数据的R值为22%（PDB代码：1 J2 G）。四面体界面环的主链结构与A.在改变盐浓度时B UOX晶体质量的不稳定性阻止了制备黄色衍生物。然而，bUOX的另一种盐效应被发现，其热稳定性随着盐浓度的增加而增加。有趣的是，在四聚体界面的环之一包含R298和硫酸根离子似乎桥接与非晶体学双重对称相关的亚基。

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。