METHANOCALDOCOCCUS JANNASCHII COBY (MJ1117)

甲烷热球菌 JANNASCHII COBY (MJ1117)

• 批准号: 8361156
• 负责人: JORGE C ESCALANTE
• 金额: $ 1.12万
• 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/8361156
• 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 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.

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