Purification and characterization of CobT, the nicotinate-mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase enzyme from Salmonella typhimurium LT2

J Biol Chem. 1997 Jul 11;272(28):17662-7. doi: 10.1074/jbc.272.28.17662.


We report the purification and biochemical characterization of the cobalamin biosynthetic enzyme nicotinate-mononucleotide:5, 6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium. cobT was overexpressed and the protein purified to approximately 97% homogeneity. NH2-terminal sequence analysis confirmed that the protein encoded by cobT was purified. Homogeneous CobT catalyzed the synthesis of N1-(5-phospho-alpha-D-ribosyl)-5,6-dimethylbenzimidazole. The identity of high performance liquid chromatography-purified product was confirmed by fast atom bombardment mass spectrometry. CobT activity was optimal at 45 degrees C and pH 10.0. The apparent Km for nicotinate mononucleotide was 680 microM; the apparent Km for 5, 6-dimethylbenzimidazole was less than 10 microM. CobT used nicotinamide mononucleotide as a ribose phosphate donor. CobT phosphoribosylated alternative base substrates including benzimidazole, 4,5-dimethyl-1,2-phenylenediamine, imidazole, histidine, adenine, and guanine in vitro. The resulting ribotides were incorporated into cobamides that were differentially utilized by methionine synthase (EC, ethanolamine ammonia-lyase (EC, and 1,2-propanediol dehydratase (EC in vivo. The lack of base substrate specificity by CobT may explain the inability to isolate mutants blocked in the synthesis of 5, 6-dimethylbenzimidazole in this bacterium.

Publication types

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

MeSH terms

  • Isoelectric Point
  • Kinetics
  • Models, Chemical
  • Multienzyme Complexes*
  • Nucleotidyltransferases*
  • Pentosyltransferases / isolation & purification*
  • Pentosyltransferases / metabolism
  • Ribosemonophosphates / metabolism
  • Salmonella typhimurium / enzymology*
  • Substrate Specificity


  • Multienzyme Complexes
  • Ribosemonophosphates
  • Pentosyltransferases
  • nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase
  • Nucleotidyltransferases