Analysis of the adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase (CobU) enzyme of Salmonella typhimurium LT2. Identification of residue His-46 as the site of guanylylation

J Biol Chem. 2000 Sep 8;275(36):27576-86. doi: 10.1074/jbc.M000977200.


CobU is a bifunctional enzyme involved in adenosylcobalamin (coenzyme B(12)) biosynthesis in Salmonella typhimurium LT2. In this bacterium, CobU is the adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase needed to convert cobinamide to adenosylcobinamide-GDP during the late steps of adenosylcobalamin biosynthesis. The guanylyltransferase reaction has been proposed to proceed via a covalently modified CobU-GMP intermediate. Here we show that CobU requires a nucleoside upper ligand on cobinamide for substrate recognition, with the nucleoside base, but not the 2'-OH group of the ribose, being important for this recognition. During the kinase reaction, both the nucleotide base and the 2'-OH group of the ribose are important for gamma-phosphate donor recognition, and GTP is the only nucleotide competent for the complete nucleotidyltransferase reaction. Analysis of the ATP:adenosylcobinamide kinase reaction shows CobU becomes less active during this reaction due to the formation of a covalent CobU-AMP complex that holds CobU in an altered conformation. Characterization of the GTP:adenosylcobinamide-phosphate guanylyltransferase reaction shows the covalent CobU-GMP intermediate is on the reaction pathway for the generation of adenosylcobinamide-GDP. Identification of a modified histidine and analysis of cobU mutants indicate that histidine 46 is the site of guanylylation.

Publication types

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

MeSH terms

  • Binding Sites
  • Chromatography, Affinity
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Cobamides / biosynthesis
  • Histidine*
  • Kinetics
  • Multienzyme Complexes / chemistry
  • Multienzyme Complexes / isolation & purification
  • Multienzyme Complexes / metabolism*
  • Mutagenesis, Site-Directed
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Salmonella typhimurium / enzymology*
  • Substrate Specificity


  • Cobamides
  • Multienzyme Complexes
  • Recombinant Proteins
  • adensoylcobinamide
  • Histidine