Publications

• Capture of a labile substrate by expulsion of water molecules from the active site of nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella enterica.

  Cheong C.G., Escalante-Semerena J.C., Rayment I.

  Nicotinate mononucleotide (NaMN):5,6-dimethylbenzimidazole (DMB) phosphoribosyltransferase (CobT) from Salmonella enterica plays a central role in the synthesis of alpha-ribazole-5'-phosphate, an intermediate for the lower ligand of cobalamin. In earlier studies it proved difficult to obtain the s ... >> More

  Nicotinate mononucleotide (NaMN):5,6-dimethylbenzimidazole (DMB) phosphoribosyltransferase (CobT) from Salmonella enterica plays a central role in the synthesis of alpha-ribazole-5'-phosphate, an intermediate for the lower ligand of cobalamin. In earlier studies it proved difficult to obtain the structure of CobT bound to NaMN because it is hydrolyzed in the crystal lattice in the absence of the second substrate DMB. In an effort to map the reaction pathway of this enzyme, NaMN was captured in the active site with the substrate analogs 4,5-dimethyl-1,2-phenylenediamine, 4-methylcatechol, indole, 3,4-dimethylaniline, 2,5-dimethylaniline, 3,4-dimethylphenol, and 2-amino-p-cresol. Structures of these complexes reveal that they exclude water molecules responsible for the hydrolysis from the active site. These structures, together with the early complexes with alpha-ribazole-5'-phosphate and DMB, provide a complete description of the reaction pathway. They demonstrate that the nicotinate moiety and phosphate do not appear to move significantly between reactants and products but that the aromatic base and ribose moiety each move approximately 1.2 A toward each other in the transformation. This study also reveals that, like many other nucleotide binding proteins, coordination of DMB is accompanied by a disorder-order transition in a surface loop. The structure of apo-CobT is also reported. << Less

  J Biol Chem 277:41120-41127(2002) [PubMed] [EuropePMC]

• Structural investigation of the biosynthesis of alternative lower ligands for cobamides by nicotinate mononucleotide: 5,6-dimethylbenzimidazole phosphoribosyltransferase from Salmonella enterica.

  Cheong C.G., Escalante-Semerena J.C., Rayment I.

  Nicotinate mononucleotide (NaMN):5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella enterica plays a central role in the synthesis of alpha-ribazole, a key component of the lower ligand of cobalamin. Surprisingly, CobT can phosphoribosylate a wide range of aromatic substrat ... >> More

  Nicotinate mononucleotide (NaMN):5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella enterica plays a central role in the synthesis of alpha-ribazole, a key component of the lower ligand of cobalamin. Surprisingly, CobT can phosphoribosylate a wide range of aromatic substrates, giving rise to a wide variety of lower ligands in cobamides. To understand the molecular basis for this lack of substrate specificity, the x-ray structures of CobT complexed with adenine, 5-methylbenzimidazole, 5-methoxybenzimidazole, p-cresol, and phenol were determined. Furthermore, adenine, 5-methylbenzimidazole, 5-methoxybenzimidazole, and 2-hydroxypurine were observed to react with NaMN within the crystal lattice and undergo the phosphoribosyl transfer reaction to form product. Significantly, the stereochemistries of all products are identical to those found in vivo. Interestingly, p-cresol and phenol, which are the lower ligand in Sporomusa ovata, bound to CobT but did not react with NaMN. This study provides a structural explanation for how CobT can phosphoribosylate most of the commonly observed lower ligands found in cobamides with the exception of the phenolic lower ligands observed in S. ovata. This is accomplished with minor conformational changes in the side chains that constitute the 5,6-dimethylbenzimidazole binding site. These investigations are consistent with the implication that the nature of the lower ligand is controlled by metabolic factors rather by the specificity of the phosphoribosyltransferase. << Less

  J. Biol. Chem. 276:37612-37620(2001) [PubMed] [EuropePMC]