title: 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.
abstract: 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.
authors.affiliation: Institute for Enzyme Research, Department of Biochemistry, University of Wisconsin, Madison 53705, USA.
authors.forename: C G
authors.initials: CG
authors.lastname: Cheong
authors.forename: J C
authors.initials: JC
authors.lastname: Escalante-Semerena
authors.forename: I
authors.initials: I
authors.lastname: Rayment
mesh_terms: D001562:Benzimidazoles / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D046911:Macromolecular Substances; D008958:Models, Molecular; D009097:Multienzyme Complexes; D009525:Niacin / Q000737:chemistry / Q000378:metabolism; D009713:Nucleotidyltransferases; D010430:Pentosyltransferases / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012265:Ribonucleotides / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D001562:Benzimidazoles; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D012265:Ribonucleotides; D009525:Niacin; C015158:5,6-dimethylbenzimidazole; C087731:N(1)-(5-phosphoribosyl)-5,6-dimethylbenzimidazole; D010430:Pentosyltransferases; C028015:nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase; D009713:Nucleotidyltransferases