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

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 N-1-(5-phospho-alpha-D-ribosyl)-5,6-dimethyl benzimidazole, 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 K-m for nicotinate mononucleotide was 680 mu M; the apparent K-m for 5,6-dimethylbenzimidazole was less than 10 mu M. 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 2,1,1.13) ethanolamine ammonia-lyase (EC 4,3.1.7), and 1,2-propanediol dehydratase (EC 4.2,1.28) 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.