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 N1-(5-phospho-alpha-D-ribosyl)-5,6-dimethylbenzimidazole. 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 Km for nicotinate mononucleotide was 680 microM; the apparent Km for 5, 6-dimethylbenzimidazole was less than 10 microM. 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 184.108.40.206), ethanolamine ammonia-lyase (EC 220.127.116.11), and 1,2-propanediol dehydratase (EC 18.104.22.168) 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.