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Volume 272, Number 28, Issue of July 11, 1997 pp. 17662-17667
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Purification and Characterization of CobT, the Nicotinate-mononucleotide:5,6-Dimethylbenzimidazole Phosphoribosyltransferase Enzyme from Salmonella typhimurium LT2

(Received for publication, February 4, 1997, and in revised form, April 30, 1997)

From the Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706-1567

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--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 °C and pH 10.0. The apparent K_m for nicotinate mononucleotide was 680 µM; the apparent K_m for 5,6-dimethylbenzimidazole was less than 10 µM. 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.

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