COMMENTARY

Finding the final pieces of the vitamin B12 biosynthetic jigsaw

Martin J. Warren* Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, United Kingdom

ver since Dorothy Hodgkin gate anaerobe Eubacterium limosum min B12 deficiency in the photosynthetic solved the structure of vitamin (13). Here, labeling revealed that the bacterium Rhodobacter capsulatus (18). B12 some 50 years ago, research- DMB framework was synthesized anaer- Campbell et al. (1) demonstrate that ers have been puzzling over how obically from erythrose, glycine, for- their S. meliloti bluB mutant strain is thisE amazing molecular jigsaw is pieced mate, glutamine, and methionine. also a B12 auxotroph but, more specifi- together. In essence, vitamin B12 is However, as with the synthesis of the cally, that it is deficient in the biosyn- a modified tetrapyrrole (corrinoid ring) corrin ring, it also was noted that some thesis of DMB. There appears to be to which is attached either an upper ad- organisms synthesized DMB aerobically a certain amount of serendipity in the enosyl or methyl group and a lower and required molecular oxygen to allow isolation of their bluB strain, because base, usually dimethylbenzimidazole its synthesis (14). In the aerobic path- there is no obvious connection between (DMB) (Fig. 1). Although significant way, it was shown that riboflavin is a requirement for B12 and the produc- success has been achieved toward an transformed into DMB through FMN tion of succinyloglycan. The blu prefix understanding of the construction of the (15) (Fig. 1). This amazing transforma- refers to the fact that the blu genes are corrin ring component of the coenzyme, tion, for which no precedent exists in required to make an aerobic culture of there has been a paucity of information chemistry, sees the C-2 carbon of DMB R. capsulatus blush (i.e., produce photo- concerning the biosynthesis of DMB. In derived from the C1 of the ribose moi- synthetic pigments) after reduction of a recent issue of PNAS, Campbell et al. ety of FMN (16, 17). Significantly, how- the pO2. This earlier research demon- (1) identified an enzyme that appears to ever, no genes or enzymes involved in strated that a bluB strain could be cor- play a key role in the transformation of either the aerobic or anaerobic biosyn- rected by the addition of vitamin B12 flavin into DMB. thesis of DMB have been identified but not by cobinamide (i.e., vitamin B12 missing the lower base). This finding Vitamin B12 is best known as the anti- so far. pernicious anemia factor, which was first suggests two things: (i) that bluB is identified 80 years ago in crude liver somehow involved in either the synthesis extracts (2) before its subsequent purifi- There appears of DMB or its attachment to the corrin cation in 1948 (3) and structure deter- ring framework, and (ii) that the biosyn- mination in 1955 (4). There are two to be a certain amount thesis of vitamin B12 somehow disturbs the biosynthesis of bacteriochlorophyll. features that make vitamin B12 stand out in comparison with all other nutri- of serendipity in In the case of the latter, strong evidence ents. First, it is made only by microbes was subsequently presented to demon- (5, 6) (seemingly its biosynthetic soft- the isolation of strate that vitamin B12 may well be ware never made the prokaryotic to required for the biosynthesis of the iso- eukaryotic transition), and second, there Campbell et al.’s cyclic ring of bacteriochlorophyll (19). is the sheer complexity of its synthesis, Perhaps we should not be surprised to which requires Ϸ30 enzyme-mediated bluB strain. find that BluB is involved in DMB bio- steps (7). The latter part of the 20th synthesis, because evidence that BluB century saw a major effort to elucidate was involved in DMB biosynthesis was the biosynthesis of this remarkable co- Campbell et al. (1) were not actually presented in a patent application, which claimed that overproduction of BluB led factor. A multidisciplinary approach trying to investigate B12 biosynthesis. involving microbiology, genetics, recom- They were studying aspects of polysac- to raised levels of DMB (20). This find- binant DNA technology, NMR, and charide synthesis in Rhizobia, the soil ing is significant because DMB is nor- chemistry saw the dissection of this bacteria that live in specialized root mally limiting in the biosynthesis of vitamin B . Thus, before the work by complex metabolic pathway (8–10). Of nodules of leguminous plants. As part of 12 Campbell et al. (1), there was evidence course, things were more complicated their symbiotic relationship with the that BluB was required for the aerobic than anticipated. It was found that there plant, the bacteria produce fixed nitro- synthesis of the lower axial base. How- were two distinct pathways for cobal- gen in return for carbon compounds. ever, what Campbell et al. were able to amin biosynthesis, representing aerobic During the invasion of the plant nod- do was to show that a mutant in bluB and anaerobic routes (11). Although the ules, the bacteria produce an acidic was able to grow in the presence of aerobic route for corrin ring assembly exopolysaccharide, termed succinogly- either vitamin B or DMB but not in was elucidated, the secrets of the anaer- can. They devised a rapid genetic screen 12 the presence of cobinamide. They also obic process have yet to be determined. for Sinorhizobium meliloti that allowed showed that bluB mutants accumulated them to identify bacteria that were ei- The attachment of the lower nucleotide GDP–cobinamide, the immediate pre- ther deficient in the production of succi- loop to the corrin ring also has been cursor of vitamin B . well established (12). However, despite noglycan or able to accumulate this 12 all this research, little information was high-molecular-weight polymer. One of forthcoming on the synthesis of the the variants from their screen that accu- Conflict of interest statement: No conflicts declared. lower nucleotide base, DMB. mulated succinylgylcan was found to See companion article on page 4634 in issue 12 of volume The majority of early work on the be inactivated in a gene that displayed 103. synthesis of the DMB moiety of vitamin a high level of similarity to bluB, a gene *E-mail: [email protected]. B12 was based on studies with the obli- previously identified as causing a vita- © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601030103 PNAS ͉ March 28, 2006 ͉ vol. 103 ͉ no. 13 ͉ 4799–4800 Downloaded by guest on September 27, 2021 Fig. 1. Transformation of FMN into DMB and its subsequent incorporation into vitamin B12. The portion of FMN that is transformed into DMB is highlighted in blue. This transformation is likely to require BluB and molecular oxygen. The incorporation of DMB into vitamin B12 requires the actions of three enzymes, CobT, C, and S.

Although the evidence is very strong biochemical proof is required to demon- process suggests a retro-aldol condensa- in supporting a role for BluB in the bio- strate that it catalyzes a reaction associ- tion sandwiched between two 2-electron synthesis of DMB, a note of caution has ated with DMB formation. Because oxidations (13, 16). This number of to be added. It had been suggested that BluB belongs to the NADH͞FMN oxi- steps seems too much to be catalyzed by CobT (NaMN:DMB phosphoribosyl- doreductase family, it is plausible that it a single (small, 15-kDa) enzyme. So are transferase) synthesized DMB (21), could catalyze the oxygenation of FMN, other enzymes involved, such as the poign- although this hypothesis was subse- a prerequisite to the fragmentation of antly named bluF gene (23)? And what quently shown not to be the case. More- the flavin. The abiotic conversion of does BluB actually do? Moreover, some over, within the B12 pathway, mutational FMN to DMB has been shown (16, 17), organisms such as Salmonella enterica inactivation of one gene sometimes can although, contrary to the suggestion in also make DMB from FMN but do not apparently affect other parts of the Campbell et al. (1), it was never claimed appear to have a bluB orthologue, so pathway. So, for instance, the activity of that that this abiotic process is the phys- how do they catalyze this reaction? CbiD (a methyltransferase) requires the iological source of DMB. Moreover, it is These are the really interesting ques- presence of the amidases CbiA and P not clear whether the transformation of tions that need to be addressed next as (22). Thus, to confirm truly the role of FMN into DMB will require one or sev- the enigmatic and furtive biosynthesis of BluB in the biosynthesis of DMB, direct eral steps (Fig. 1). The chemistry of the cobalamin is further unraveled.

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