Pseudo-B 12 Joins the Cofactor Family (cid:1)

Over 50 years ago, a deadly autoimmune disease known as pernicious anemia, which is characterized by neurological and hematological abnormalities, had no known cure. The isolation of the anti-pernicious anemia factor from liver extracts by Folkers and Smith in 1948 represented a breakthrough in the treatment of this disease (21, 25). Named vitamin B12, this compound could be isolated as pure, red crystals from various animal and microbial sources, and the X-ray crystal structure of this complex molecule, one of the largest nonpolymeric natural compounds, was determined in the 1950s by Dorothy Hodgkin et al. (Fig. 1A) (8). Since that time, thousands of researchers have identified and characterized a variety of enzymes that utilize B12 cofactors, determined the complex pathway for B12 absorption in humans, and elucidated the microbial pathways for B12 biosynthesis. Other forms of vitamin B12 have been found in nature, but their functions are not fully understood. In this issue of the Journal of Bacteriology, Anderson et al. provide evidence that a form of B12 known as “pseudoB12” (Fig. 1B) is a physiologically important form of B12 in Salmonella enterica serovar Typhimurium (1). Unlike all other vitamins, vitamin B12 is not produced by plants; rather, its biosynthesis is restricted to certain prokaryotes (23). Animals obtain B12 from food or, in some cases, directly from resident B12-producing microbes (28). S. enterica has long been used as a model organism for studying B12 biosynthesis (23). B12 structure and biosynthesis. B12 and B12-like compounds are collectively termed corrinoids. The structural core of corrinoids is the corrin ring in which the central cobalt atom is coordinated by four pyrrole groups (Fig. 1) (30). Two axial ligands also coordinate the cobalt atom. In the cofactor forms, the upper ( ) axial ligand (R) consists of either 5 -deoxyadenosine or a methyl group, which facilitate radical or methyl transfer reactions, respectively (14). The lower ( ) axial ligand is attached to the corrin ring via the nucleotide loop which is characterized by an unusual -glycosidic linkage to C-1 of the ribosyl moiety (30). The B12 cofactors found in animals and many bacteria contain 5,6-dimethylbenzimidazole (DMB) as the -axial ligand (Fig. 1A; B12 with DMB will be abbreviated hereafter as B12[DMB] to distinguish it from pseudo-B12) (26). B12 cofactors with -axial ligands other than DMB have also been observed, with the most common example being pseudoB12 (Fig. 1B), in which N7-linked adenine replaces DMB as the -axial ligand (9). The complete biosynthesis of vitamin B12 requires 30 gene products. The B12 biosynthetic pathway is characterized by two converging branches: the synthesis of the corrin ring and the synthesis and activation of the -axial ligand (23, 30). Two pathways for corrin ring synthesis have been discovered and are hypothesized to be derived from a common ancestor (10). The main difference between these pathways is in the participation of oxygen. The “aerobic” pathway requires oxygen and has been characterized in depth in Pseudomonas denitrificans (7, 30). The “anaerobic” pathway, characterized in the aerotolerant anaerobe Propionibacterium shermanii and the facultative anaerobe S. enterica, is sensitive to oxygen, and thus, the expression of B12 biosynthetic enzymes is restricted to anaerobic or microaerobic conditions (22, 30). Nucleotide loop assembly is similar in both pathways (23, 30). Two independent pathways for DMB synthesis also exist (18). Aerobic DMB synthesis occurs by fragmentation of a flavin cofactor and is catalyzed by the BluB enzyme (6, 19, 27). 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). Probing the origin of the -axial ligand of B12 in S. enterica. The observation that S. enterica produces B12[DMB], but that no DMB biosynthetic enzyme has been identified in S. enterica, has puzzled researchers for many years (23). Anderson et al. explored this issue by selecting for mutants of S. enterica that grow under conditions that normally require exogenous DMB (1). One key observation that made this selection possible is that traditional agar contains traces of DMB, and thus, much of the DMB in the B12[DMB] produced by S. enterica can be attributed to its presence in agar. Using purified agar enabled the authors to study only the endogenously produced corrinoids. 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 B12 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). 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-B12 (1). Thus, all of the characterized mutants pro* Corresponding author. Mailing address: Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139. Phone: (617) 253-6716. E-mail: gwalker@mit . edu. Published ahead of print on 14 December 2007.