One Pathway Can Incorporate either Adenine or Dimethylbenzimidazole as an α-Axial Ligand of B ₁₂ Cofactors in Salmonella enterica

Abstract

Coenzyme B 12 (Ado-Cbl) and vitamin B 12 (CN-B 12 ) (Fig. 1) include the base 5,6-dimethylbenzimidazole (DMB) as an ␣-axial ligand. Prokaryotes, the only producers of vitamin B 12 , make "complete" corrinoid cofactors with a variety of alternative ␣-axial ligands, including benzimidazoles, phenols, and purines (16, 52). These alternative corrinoids may be functional equivalents of coenzyme B 12 for many bacterial enzymes, but functional differences are suggested by the selectivity of the human assimilatory protein, intrinsic factor for cobalamin, the DMB-containing vitamin B 12 (53).Under strictly anaerobic growth conditions, Salmonella enterica synthesizes corrinoids de novo (44) and installs either adenine (to form pseudo-coenzyme B 12 [Ado-pseudo-B 12 ]) or 2-methyl-adenine (to form adenosyl-factor A) as an ␣-axial ligand (22). If even trace amounts of oxygen are present, DMB-containing B 12 (cobalamin [Cbl]) is also made (39). When grown aerobically on glucose, S. enterica cannot synthesize the corrin moiety, which must be supplied as an "incomplete" corrinoid, such as cobinamide (Cbi), with its corrin ring and aminopropanol side chain. Under these conditions, coenzyme B 12 is made, with DMB as the ␣-axial ligand (20). Figure 2 diagrams the de novo (anaerobic) synthetic pathway and assimilation of exogenous Cbi or . Notice that the ␤-ligand adenosyl can be added (by CobA) to three different substrates and that the (CN) 2 Cbi supplied as the corrin ring source is not a normal CobA substrate.Here, the origins and installation of ␣-axial ligands are approached genetically using an unexpected feature of S. enterica. While S. enterica can use exogenous Cbi to produce B 12 cofactors (cobalamins) during aerobic growth on glucose (20), it makes only about 100 molecules per cell (2). This is apparently insufficient B 12 coenzyme to support growth on ethanolamine (5) unless the DMB base is also supplied. That is, under these conditions, cells neither make sufficient DMB nor install an alternative ligand to allow corrinoid-dependent aerobic growth on ethanolamine (5).This situation allowed positive selection of mutants that can grow on ethanolamine plus Cbi without added DMB. These mutants were expected to show either increased endogenous DMB production or to install an alternative base as an ␣-axial ligand. All of the mutants made pseudo-B 12 cofactors, which have adenine as an ␣-axial ligand, and most of these mutations affected purine metabolism so as to increase the intracellular level of free adenine base. The same set of enzymes (CobUSTC) installs either adenine base (to form pseudo-B 12 ) or DMB (to form vitamin B 12 ). A model suggests how the choice is made. MATERIALS AND METHODSBacterial strains and transposons. Strains were derived from S. enterica (serovar Typhimurium) LT2

Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis

“…1C), resulting in a functional AdoCbl analog. Previous studies in Salmonella enterica, for example, found point mutations causing incorporation of adenine in place of dimethylbenzimidazole, resulting in formation of pseudo-B 12 (58). An alternative explanation is that the spontaneous mutation in some way causes increased anaerobiosis or increased resistance of NrdD to O 2 .…”

Pseudo-B 12 Joins the Cofactor Family

“…Unexpectedly, rather than uncovering genes involved in synthesizing DMB, the authors identified 25 point mutations that increase the cellular concentration of free adenine, a precursor of pseudo-B 12 (1). Thus, all of the characterized mutants pro-duced an elevated level of pseudo-B 12 that supported growth on ethanolamine.…”

“…Another important difference between this and other studies was that growth was monitored on ethanolamine rather than methionine or propanediol. This alteration was critical because (i) the requirement for B 12 during growth on ethanolamine is more stringent than on methionine, and (ii) exogenous DMB is required for growth on ethanolamine but not on propanediol (1).…”

“…DMB synthesis in some anaerobes appears to branch from the purine biosynthetic pathway, although enzymes involved in this process have not been isolated (11,18). Interestingly, S. enterica synthesizes the corrin ring anaerobically and derives DMB aerobically from flavin mononucleotide (FMN), although no ortholog of BluB appears to exist in the genome (1,12).…”

Two distinct pools of B12analogs reveal community interdependencies in the ocean

“…Rhodobacters have the O 2 -dependent corrin ring and DMB pathways (11), whereas Thaumarchaeota likely possess the O 2 -independent pathway for the corrin ring and the O 2 -dependent DMB pathway (10). In some bacteria, pseudocobalamin can be produced if DMB synthesis is impaired; this is due to the natural presence of adenine in cells and enzyme substrate promiscuity that allows adenine to substitute for DMB in some organisms' CobT (22)(23)(24)(25)(26)(27). Cyanobacteria use the O 2 -independent pathway for corrin ring synthesis and neither pathway for DMB synthesis (11,18) (Fig.…”