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

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

Author information

1
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.
2
Department of Microbiology, University of Georgia, Athens, Georgia, United States of America.
3
National Magnetic Resonance Facility at Madison and Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

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.

PMID:
26513744
PMCID:
PMC4626045
DOI:
10.1371/journal.pone.0141297
[Indexed for MEDLINE]
Free PMC Article

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