Solution Structural Studies of GTP:Adenosylcobinamide-Phosphateguanylyl Transferase (CobY) from Methanocaldococcus jannaschii

Kiran K. Singarapu, Michele M. Otte, Marco Tonelli, William M. Westler, Jorge C. Escalante-Semerena, John L. Markley, Eugene A. Permyakov
2015 PLoS ONE  
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
more » ... ann 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 1 H-15 N 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 CobY G153D , 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 CobY G153D :GTP complex was also found to be unstable over time.
doi:10.1371/journal.pone.0141297 pmid:26513744 pmcid:PMC4626045 fatcat:3twfuiktobc2rmmziy3iglftwy