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

PLoS One. 2015 Oct 29;10(10):e0141297. doi: 10.1371/journal.pone.0141297. eCollection 2015.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Guanosine Triphosphate / chemistry*
  • Guanosine Triphosphate / metabolism
  • Ligands
  • Methanocaldococcus / metabolism*
  • Models, Molecular*
  • Molecular Conformation*
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / metabolism
  • Nuclear Magnetic Resonance, Biomolecular
  • Nucleotidyltransferases / chemistry*
  • Nucleotidyltransferases / metabolism
  • Pentosyltransferases / chemistry*
  • Pentosyltransferases / metabolism
  • Protein Binding
  • Quantitative Structure-Activity Relationship
  • Solutions

Substances

  • Ligands
  • Multienzyme Complexes
  • Solutions
  • Guanosine Triphosphate
  • Pentosyltransferases
  • nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase
  • Nucleotidyltransferases