title: Solution Structural Studies of GTP:Adenosylcobinamide-Phosphateguanylyl Transferase (CobY) from Methanocaldococcus jannaschii.
abstract: GTP:adenosylcobinamide-phosphate (AdoCbi-P) guanylyl transferase (CobY) is an enzyme that transfers the GMP moiety of GTP to AdoCbi yielding AdoCbi-GDP in the late steps of the assembly of Ado-cobamides in archaea. The failure of repeated attempts to crystallize ligand-free (apo) CobY prompted us to explore its 3D structure by solution NMR spectroscopy. As reported here, the solution structure has a mixed α/β fold consisting of seven β-strands and five α-helices, which is very similar to a Rossmann fold. Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation. However, the CobY:GTP complex as followed by 1H-15N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY. The variant CobYG153D, whose GTP complex was studied by X-ray crystallography, yielded NMR spectra similar to those of wild-type CobY in both its apo- state and in complex with GTP. The CobYG153D:GTP complex was also found to be unstable over time.
authors.affiliation: National Magnetic Resonance Facility at Madison and Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America; Center for NMR and Structural Chemistry, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, Telangana, India.
authors.forename: Kiran K
authors.initials: KK
authors.lastname: Singarapu
authors.affiliation: Department of Microbiology, University of Georgia, Athens, Georgia, United States of America.
authors.forename: Michele M
authors.initials: MM
authors.lastname: Otte
authors.affiliation: National Magnetic Resonance Facility at Madison and Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Marco
authors.initials: M
authors.lastname: Tonelli
authors.affiliation: National Magnetic Resonance Facility at Madison and Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: William M
authors.initials: WM
authors.lastname: Westler
authors.affiliation: Department of Microbiology, University of Georgia, Athens, Georgia, United States of America.
authors.forename: Jorge C
authors.initials: JC
authors.lastname: Escalante-Semerena
authors.affiliation: National Magnetic Resonance Facility at Madison and Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: John L
authors.initials: JL
authors.lastname: Markley
mesh_terms: D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D063747:Methanocaldococcus / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D010430:Pentosyltransferases / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D021281:Quantitative Structure-Activity Relationship; D012996:Solutions
chemical_list: D008024:Ligands; D009097:Multienzyme Complexes; D012996:Solutions; D006160:Guanosine Triphosphate; D010430:Pentosyltransferases; C028015:nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase; D009713:Nucleotidyltransferases