Purification and characterization of the bifunctional CobU enzyme of Salmonella typhimurium LT2. Evidence for a CobU-GMP intermediate

J Biol Chem. 1995 Oct 6;270(40):23560-9. doi: 10.1074/jbc.270.40.23560.


The CobU protein of Salmonella typhimurium was overexpressed and purified to approximately 94% homogeneity. N-terminal sequencing of purified CobU confirmed the first 22 amino acids. In vitro assays showed that CobU has kinase and guanylyltransferase activities which catalyze the synthesis of adenosyl-cobinamide-GDP from adenosyl-cobinamide, via an adenosyl-cobinamide-phosphate intermediate. We present evidence that the transfer of the guanylyl moiety of GTP to adenosyl-cobinamide-phosphate proceeds via an phosphoramidate-linked, enzyme-guanylyl intermediate. In the presence of oxygen, kinase and guanylyltransferase activities of CobU were lost. Treatment of inactive CobU with dithiothreitol restored approximately 20% of the kinase and guanylyltransferase activities, indicating the involvement of sulfhydryl groups in enzyme activity. The sulfhydryl modifying agents 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide abolished both CobU activities. Native CobU protein was a dimer (approximately 40 kDa) that functioned optimally at pH 8.8-9.0 and 37 degrees C. Substrates and kinetic parameters for both activities were determined. The preferred corrinoid substrate for this enzyme was adenosyl-cobinamide. In vitro experiments are consistent with previous genetic studies which had suggested that adenosyl-cobinamide was the preferred substrate of CobU, and that CobU functioned more efficiently in the absence of oxygen.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cobamides / metabolism
  • DNA, Bacterial / genetics
  • Dithionitrobenzoic Acid / pharmacology
  • Enzyme Stability
  • Gene Expression
  • Genes, Bacterial
  • Guanosine Monophosphate / metabolism
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Sequence Data
  • Multienzyme Complexes*
  • Mutagenesis, Site-Directed
  • Nucleotidyltransferases*
  • Pentosyltransferases / genetics
  • Pentosyltransferases / isolation & purification*
  • Pentosyltransferases / metabolism
  • Protein Conformation
  • Salmonella typhimurium / enzymology*
  • Salmonella typhimurium / genetics
  • Substrate Specificity


  • Cobamides
  • DNA, Bacterial
  • Multienzyme Complexes
  • Guanosine Monophosphate
  • Dithionitrobenzoic Acid
  • Pentosyltransferases
  • nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase
  • Nucleotidyltransferases