METHANOCALDOCOCCUS JANNASCHII COBY (MJ1117)

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 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. 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.