The three-dimensional structures of nicotinate mononucleotide:5,6- dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium complexed with 5,6-dimethybenzimidazole and its reaction products determined to 1.9 A resolution

Biochemistry. 1999 Dec 7;38(49):16125-35. doi: 10.1021/bi991752c.


Nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium plays a central role in the synthesis of alpha-ribazole, which is a key component of the lower ligand of cobalamin. Two X-ray structures of CobT are reported here at 1.9 A resolution. First, a complex of CobT with 5,6-dimethylbenzimidazole, and second, a complex of CobT with its reaction products, nicotinate and alpha-ribazole-5'-phosphate. CobT was cocrystallized with 5,6-dimethylbenzimidazole (DMB) in the space group P2(1)2(1)2 with unit cell dimensions of a = 72.1 A, b = 90.2 A, and c = 47.5 A and one protomer per asymmetric unit. Subsequently, the crystals containing DMB were soaked in nicotinate mononucleotide whereupon the physiological reaction occurred in the crystal lattice to yield nicotinate and alpha-ribazole-5'-phosphate. These studies show that CobT is a dimer where each subunit consists of two domains. The large domain is dominated by a parallel six-stranded beta-sheet with connecting alpha-helices that exhibit the topology of a Rossmann fold. The small domain is made from components of the N- and C-terminal sections of the polypeptide chain and contains a three-helix bundle. The fold of CobT is unrelated to the type I and II phosphoribosylpyrophosphate dependent transferases and does not appear to be related to any other protein whose structure is known. The enzyme active site is located in a large cavity formed by the loops at the C-terminal ends of the beta-strands and the small domain of the neighboring subunit. DMB binds in a hydrophobic pocket created in part by the neighboring small domain. This is consistent with the broad specificity of this enzyme for aromatic substrates [Trzebiatowski, J. R., Escalante-Semerena (1997) J. Biol. Chem. 272, 17662-17667]. The binding site for DMB suggests that Glu317 is the catalytic base required for the reaction. The remainder of the cavity binds the nicotinate and ribose-5'-phosphate moieties, which are nestled within the loops at the ends of the beta-strands. Interestingly, the orientation of the substrate and products are opposite from that expected for a Rossmann fold.

Publication types

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

MeSH terms

  • Benzimidazoles / chemistry*
  • Benzimidazoles / metabolism
  • Binding Sites
  • Catalysis
  • Crystallization
  • Crystallography, X-Ray
  • Macromolecular Substances
  • Models, Molecular
  • Multienzyme Complexes*
  • Niacin / chemistry
  • Niacin / metabolism
  • Nucleotidyltransferases*
  • Pentosyltransferases / chemistry*
  • Pentosyltransferases / metabolism
  • Phosphorylation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Ribonucleotides / chemistry
  • Ribonucleotides / metabolism
  • Salmonella typhimurium / enzymology*
  • Substrate Specificity


  • Benzimidazoles
  • Macromolecular Substances
  • Multienzyme Complexes
  • Ribonucleotides
  • Niacin
  • 5,6-dimethylbenzimidazole
  • N(1)-(5-phosphoribosyl)-5,6-dimethylbenzimidazole
  • Pentosyltransferases
  • nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase
  • Nucleotidyltransferases

Associated data

  • PDB/1D0S
  • PDB/1D0V