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-5,6-Dimethylbenzimidazole adenine dinucleotide (-DAD), a putative new intermediate of coenzyme B₁₂ biosynthesis in Salmonella typhimurium

Department of Bacteriology, University of Wisconsin, 1710 University Avenue, Madison, WI 53726-4087, USA

Correspondence
Jorge C. Escalante-Semerena
escalante{at}bact.wisc.edu

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 -5,6-dimethylbenzimidazole adenine dinucleotide (-DAD), whose identity was established by mass spectrometry. The N¹-(-D-ribosyl)-5,6-dimethylbenzimidazoyl moiety (-ribazole) of -DAD was incorporated into adenosylcobalamin (AdoCbl) by cell-free extracts of S. typhimurium, indicating that -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 -DAD into -ribazole-5'-P and AMP; -ribazole-5'-P can then enter the AdoCbl biosynthetic pathway.

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