METHANOCALDOCOCCUS JANNASCHII COBY (MJ1117)

甲烷热球菌 JANNASCHII COBY (MJ1117)

• 批准号: 8361210
• 负责人: JOHN LUTE MARKLEY
• 金额: $ 0.2万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361210
• 关键词: Anabolism; Archaea; Binding; Biochemical; Carbon; Characteristics; Cobalamin; Enzymes; Escherichia coli; Funding; Genes; Grant; Guanosine Triphosphate; Kinetics; Label; NMR Spectroscopy; National Center for Research Resources; Nitrogen; Nuclear Magnetic Resonance; Phosphotransferases; Principal Investigator; Proteins; Reporting; Research; Research Infrastructure; Resources; Salmonella enterica; Solutions; Source; Specificity; Structure; United States National Institutes of Health; base; cost; 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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Structural and Biochemical Characterization of the GTP:Adenosylcobinamide-phosphate Guanylyltransferase (CobY) Enzyme from the Hyperthermophilic Archaeon Methanocaldococcus jannaschiiThe 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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 GTP 的结构和生化特征：来自超嗜热古细菌詹氏热球菌的 CobY 酶古细菌 cobY 基因是细菌 NTP：腺苷酸鸟苷激酶/GTP：腺苷酸鸟苷酸转移酶（肠沙门氏菌中的 cobU）的非同源替换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。