n(1)-(5-phosphoribosyl)-5-6-dimethylbenzimidazole and 5-6-dimethylbenzimidazole

The CobT enzyme of Salmonella typhimurium was shown in vitro to have NAD(+)-dependent ADPribosyltransferase activity. The CobT enzyme transferred the ADPribosyl moiety of NAD(+) onto 5,6-dimethylbenzimidazole (DMB) yielding a new dinucleotide, namely alpha-5,6-dimethylbenzimidazole adenine dinucleotide (alpha-DAD), whose identity was established by mass spectrometry. The N(1)-(alpha-D-ribosyl)-5,6-dimethylbenzimidazoyl moiety (alpha-ribazole) of alpha-DAD was incorporated into adenosylcobalamin (AdoCbl) by cell-free extracts of S. typhimurium, indicating that alpha-DAD served as an intermediate of AdoCbl biosynthesis. The rate of transfer of the ADPribosyl moiety was slower than the rate of transfer of the phosphoribosyl moiety of nicotinate mononucleotide (NaMN) to DMB. The CobT enzyme displayed a low K(m) for NaMN (0.51 mM) relative to the one for NAD(+) (9 mM); nicotinate adenine dinucleotide (NaAD) and nicotinamide mononucleotide (NMN) also served as substrates for CobT. In spite of the high K(m) of CobT for NAD(+), the latter is proposed to be a relevant physiological substrate of CobT, given that the intracellular concentrations of NaMN, NMN and NaAD in actively growing S. typhimurium are undetectable. Evidence shows that extracts of S. typhimurium contain an as-yet unidentified dinucleotide pyrophosphatase that can cleave alpha-DAD into alpha-ribazole-5'-P and AMP; alpha-ribazole-5'-P can then enter the AdoCbl biosynthetic pathway.

Topics: Adenine Nucleotides; ADP Ribose Transferases; Benzimidazoles; Cobamides; Kinetics; Multienzyme Complexes; NAD; Nucleotidyltransferases; Pentosyltransferases; Ribonucleotides; Salmonella typhimurium; Substrate Specificity; Vitamin B 12

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.

Topics: Benzimidazoles; Binding Sites; Catalysis; Crystallization; Crystallography, X-Ray; Macromolecular Substances; Models, Molecular; Multienzyme Complexes; Niacin; Nucleotidyltransferases; Pentosyltransferases; Phosphorylation; Protein Structure, Secondary; Protein Structure, Tertiary; Ribonucleotides; Salmonella typhimurium; Substrate Specificity

We present in vitro evidence which demonstrates that CobT is the nicotinate nucleotide:5,6-dimethylbenzimidazole (DMB) phosphoribosyltransferase (EC 2.4.2.21) that catalyzes the synthesis of N1-(5-phospho-alpha-D-ribosyl)-5,6-dimethylbenzimidazole, a biosynthetic intermediate of the pathway that assembles the nucleotide loop of cobalamin in Salmonella typhimurium. Mutants previously isolated as DMB auxotrophs are shown by physical and genetic mapping studies and complementation studies to carry lesions in cobT. Explanations for this unexpected phenotype of cobT mutants are discussed. The expected nucleotide loop assembly phenotype of cobT mutants can be observed only in a specific genetic background, i.e., cobB deficient, an observation that is consistent with the existence of an alternative CobT function (G. A. O'Toole, M. R. Rondon, and J. C. Escalante-Semerena, J. Bacteriol. 175:3317-3326, 1993). Computer analysis of CobT homologs showed that at the amino acid level, enteric CobT proteins were 80% identical whereas Pseudomonas denitrificans and Rhizobium meliloti CobT proteins were 95% identical. Interestingly, the degree of identity between enteric and nonenteric CobT homologs was only 30%. The same pattern of homologies was reported for the S. typhimurium CobA, Escherichia coli BtuR, and P. denitrificans CobO proteins (S.-J. Suh and J.C. Escalante-Semerena, Gene 129:93-97, 1993), suggesting evolutionary divergence between the cob genes found in the enteric bacteria E. coli and S. typhimurium and those found in P. denitrificans and R. meliloti.

Topics: Amino Acid Sequence; Benzimidazoles; Chromosome Mapping; Genes, Bacterial; Molecular Sequence Data; Multienzyme Complexes; Mutation; Nucleotidyltransferases; Pentosyltransferases; Ribonucleotides; Salmonella typhimurium; Sequence Homology, Amino Acid; Vitamin B 12