METHANOCALDOCOCCUS JANNASCHII COBY (MJ1117)

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 古生型嗜热古细菌腺苷钴酰胺-磷酸鸟苷酸转移酶(Coby)的结构和生化特性古生菌的Coby基因是细菌NTP：腺苷钴酰胺/GTP(肠沙门氏菌中的CoBu)的非同源替换基因，是钴胺生物合成所必需的。我们在大肠杆菌中高效表达了简氏甲烷球菌MJ1117(Coby)基因产物(Mj Coby)，并对其进行了纯化。我们在这里报道了Mj Coby的三维结构，通过碳-13和氮-15标记的蛋白质的溶液核磁共振(核磁共振)谱确定。我们发现Mj Coby具有与Cobu相关的GTP：腺苷钴酰胺-磷酸鸟苷转移酶活性。Mj Coby的生化特性包括底物结合和专一性、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Mj Coby在合成钴胺的过程中将GTP的鸟酰基部分转移到腺苷钴酰胺-磷酸的机理。

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.