METHANOCALDOCOCCUS JANNASCHII COBY (MJ1117)

甲烷热球菌 JANNASCHII COBY (MJ1117)

• 批准号: 8168943
• 负责人: JORGE C ESCALANTE
• 金额: $ 1.36万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168943
• 关键词: Anabolism; Archaea; Binding; Biochemical; Carbon; Characteristics; Cobalamin; Computer Retrieval of Information on Scientific Projects Database; Enzymes; Escherichia coli; Funding; Genes; Grant; Guanosine Triphosphate; Institution; Kinetics; Label; NMR Spectroscopy; Nitrogen; Phosphotransferases; Proteins; Reporting; Research; Research Personnel; Resources; Salmonella enterica; Solutions; Source; Specificity; Structure; United States National Institutes of Health; base; guanylyltransferase; inorganic phosphate; overexpression; three dimensional structure

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Structural and Biochemical Characterization of the GTP:Adenosylcobinamide-phosphate Guanylyltransferase (CobY) Enzyme from the Hyperthermophilic Archaeon Methanocaldococcus jannaschii The archaeal cobY gene is the nonorthologous replacement of the bacterial NTP:adenosylcobinamide kinase/GTP:adenosylcobinamide-phosphate guanylyltransferase (cobU in Salmonella enterica) and is required for the biosynthesis of cobalamin. We overexpressed the Methanocaldococcus jannaschii MJ1117 (cobY) gene product (Mj CobY) in Escherichia coli and purified it to homogeneity. We report here the three-dimensional structure of Mj CobY, determined by solution nuclear magnetic resonance (NMR) spectroscopy from protein labeled with carbon-13 and nitrogen-15. We show that Mj CobY has the GTP:adenosylcobinamide-phosphate guanylyltransferase activity associated with CobU. The biochemical characterization of Mj CobY includes an analysis of substrate binding and specificity, physical characteristics, and kinetic parameters. Based on the Mj CobY structure and biochemical parameters, we propose a mechanism whereby Mj CobY transferse the guanylyl moiety of GTP to adenosylcobinamide-phosphate during the synthesis of cobalamin.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 来自超嗜热古菌詹纳斯基甲烷热球菌的 GTP：腺苷酸磷酸脒基转移酶 (CobY) 的结构和生化特征古菌 cobY 基因是细菌 NTP：腺苷酸酰胺的非直系同源替代物 激酶/GTP：腺苷酸鸟苷酸转移酶（肠沙门氏菌中的 cobU），是钴胺素生物合成所必需的。 我们在大肠杆菌中过表达詹氏甲烷球菌 MJ1117 (cobY) 基因产物 (Mj CobY)，并将其纯化至均质。 我们在此报告了 Mj CobY 的三维结构，该结构是通过溶液核磁共振 (NMR) 光谱从碳 13 和氮 15 标记的蛋白质中确定的。 我们表明，Mj CobY 具有与 CobU 相关的 GTP：腺苷酸酰胺-磷酸鸟苷基转移酶活性。 Mj CobY 的生化表征包括底物结合和特异性、物理特性和动力学参数的分析。 基于Mj CobY结构和生化参数，我们提出了一种机制，在钴胺素合成过程中，Mj CobY将GTP的鸟苷基部分转移到腺苷钴酰胺磷酸上。