METHANOCALDOCOCCUS JANNASCHII COBY (MJ1117)

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 超嗜热菌詹氏甲烷球菌的GTP：腺苷酸氨基-磷酸鸟苷转移酶（CobY）的结构和生化特性古细菌的cobY基因是细菌NTP：腺苷酸氨基激酶/GTP：腺苷酸氨基-磷酸鸟苷转移酶（肠道沙门氏菌中的cobU）的非邻位取代，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MJ 1117（cobY）基因产物（Mj CobY），并将其纯化至均一。 我们在这里报告的三维结构的MJ CobY，确定由溶液核磁共振（NMR）光谱从蛋白质标记的碳-13和氮-15。 我们发现，Mj CobY具有与CobU相关的GTP：腺苷可宾酰胺磷酸鸟苷转移酶活性。 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的结构和生化参数，我们提出了一种机制，即Mj CobY转移GTP的鸟苷酰部分腺苷钴胺磷酸合成钴胺素。

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 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.