COMMENTARY COMMENTARY Illuminating the black box of B12 biosynthesis Harry A. Dailey1 synthesis, one aerobic and the other anaer- Department of Microbiology, and Department of Biochemistry and Molecular Biology, obic. Whereas the genes required for both Biomedical and Health Sciences Institute, P.D. Coverdell Center, University of Georgia, Athens, aerobic and anaerobic synthesis have been GA 30602 known, the actual mechanism for synthesis via the anaerobic pathway has remained a large “black box” in what is one of the long- Tetrapyrroles are nearly ubiquitous in nature increasing or decreasing reactivity of the met- est known biosynthetic pathways. However, as participants in a wide variety of biological al center, and providing protection against the contents of this box have now been iden- reactions that are central to life, such as fi undesirable side reactions. One nds magne- tified and characterized, thanks to an elegant electron transfer, gas binding, and one-car- sium in chlorophyll, iron in hemes, nickel in study that can properly be called a tour de bon metabolism (1, 2). Because of their di- factor F430, and cobalt in cyano-cobalamin force by Moore et al. at the University of — verse colors for example the greens of plant (vitamin B12) (Fig. 1). Although hemes and Kent (3). chlorophylls, reds of blood, and blues and chlorophyls are synthesized by both prokar- Research into the metabolism of metallo- — browns of avian eggs tetrapyrroles have yotes and eukaryotes, factor F430,whichis tetrapyrroles and the biological and medical been called the “pigments of life.” The chem- involved in methanogenesis, is produced only impact of disordered synthesis or degradation ical diversity of cyclic tetrapyrroles owes by some archae, and cobalamin synthesis is of these compounds has been ongoing for the much to their ability to coordinate a variety found only in bacteria and archae. With re- past century, so that we now have a reason- of redox active metals in a fashion that al- gard to cobalamin, it has long been known able understanding of this process (1), with lows for fine-tuning of midpoint potentials, that two distinct pathways exist for its bio- the notable exceptions of cobalamin and factor F430 (4, 5). As might be expected from the related structures of tetrapyrroles, all share a common synthetic origin. The pathway’s first committed intermediate is 5-aminolevulinate, which arises from ei- ther succinyl CoA and glycine in metazo- ans, or glutamyl tRNA in most other organisms (6). The next three enzymes, porphobilinogen synthase, hydroxymethyl- bilane synthase, and uroporphyrinogen III synthase, comprise the common pathway, which is highly conserved across all species. The product of this common pathway is uroporphyrinogen III. From here, synthesis of chlorophyll and protoheme proceed to the shared precursor protoporphyrin IX via an identical route, whereas synthesis of siroheme and heme d1 use a unique and perhaps more ancient pathway in which the macrocycle is oxidized and methylated before iron insertion (7, 8). Despite its bio- logical importance in methanogenesis, factor ’ F430 s biosynthetic pathway has yet to be elu- cidated, although it shares the common path- way to uroporphyrinogen III. Given that this compound, which is a tetrahydroporphyrino- gen, is the most reduced of those previously mentioned and the only known metallo- tetrapyrrole with nickel as the chelated

Author contributions: H.A.D. wrote the paper.

The author declares no conflict of interest.

Fig. 1. The family of tetrapyrroles originating from uroporphyrinogen III. The central metal atom is colored to See companion article on page 14906. represent the color of the metallo-tetrapyrrole. 1E-mail: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1313998110 PNAS | September 10, 2013 | vol. 110 | no. 37 | 14823–14824 Downloaded by guest on October 2, 2021 metal, its biosynthesis can be anticipated to the Metazoa or prokaryotes, dietary heme is In addition to identifying black box reac- possess unique features (5). Cobalamin syn- generally broken down and the iron released tions in the anaerobic biosynthesis of co- thesis also branches off at uroporphyrinogen for reutilization, frequently to make heme. balamin, the work of Moore et al. (3) pro- III, but here one of the early steps is the Similarly, chlorophylls and factor F430 are syn- vides observations with a potentially much condensation of the porphin ring structure thesized by the organisms that possess them broader impact. First is the characterization into a corrin by elimination of one of the and are not acquired from dietary sources. of a nonredox role for cobalt in the reaction. bridging methine carbons (4). This process is different for cobalamin be- The putative role cobalt plays has not been Moore et al. tackled illumination of the cause it is synthesized only in prokaryotes; described previously in a biological system, anaerobic cobalamin synthesis black box by metazoans—which require cobalamin—can- but the current observation may prompt taking advantage of the ability to express at notsynthesizeitand,therefore,mustacquire researchers to reexamine other cobalt-con- high levels and purify all 14 pathway en- it from their diet. taining enzymes. Second is the observation zymes in soluble forms from Bacillus mega- Cobalamins are essential for three enzyme- that a mixture containing all pathway en- terium (3). This process allowed them to not catalyzed reactions: methyltransferases, iso- zymes functions more effectively than indi- only characterize each step of the black box, merases, and reductive dehalogenases (9). In vidual enzymes to produce product. This fi but also to synthesize in good yield the end humans two enzymes require cobalamin: ndingisnotunlikewhathasbeenreported product, cobyrinic acid, from the starting methylmalonyl CoA mutase, which converts forsirohemebiosynthesis(7,11)andhinted compound 5-aminolevulinate, by using a methylmalonyl CoA into succinyl CoA, and at for protoheme synthesis (12). Given the mixture of all 14 enzymes. The integration 5-methyltetrahydrofolate-homocysteine me- reactivity of tetrapyrrole biosynthetic path- of UV-Vis spectroscopy, LC-MS, and elec- way intermediates, it is not a surprise that thyltransferase (methionine synthase), which tron paramagnetic resonance allowed for evolution has designed pathway enzymes to is required for recycling of tetrahydrofolate the identification of all intermediates and hold on to products until downstream ac- via the synthesis of methionine from homo- revealed some unexpected and exciting find- ceptors are available. Such an approach, ei- cysteine. Insufficient cobalamin can result in ings about this long and complex biosyn- ther alone or with substrate channeling, may nerve myelin damage from accumulation of thetic pathway. These investigators deter- represent a generic approach to minimizing methylmalonyl CoA, and megaloblastic ane- mined that the intermediate precorrin 5B is potential toxic cellular effects of free pathway the oxygen-sensitive intermediate in the path- mia because of an inability to regenerate tet- intermediates or nonproductive loss of reac- way. This observation is a major accomplish- rahydrofolate for thymine synthesis. Dietary tive intermediates: a clear example of how ment that answers a long-standing question folate supplementation can ameliorate the Nature may have evolved complex pathways as to why this route to cobalamin is oxygen- anemia, but not myelin damage or accumu- with reactive intermediates. sensitive. Although there were previous stud- lation of homocysteine (homocysteinuria), Although the current contribution helps which may contribute to stroke and cardiac ies hinting at what may have been going on bring some closure to the B12 biosynthesis in the black box, the current data now pro- infarction. Because most nonvegetarian diets field with the illumination of the anaerobic fi vide compelling evidence and clearly settle provide a reasonable source of B12,de cien- black box components, to complement work the question. cy of cobalamin in metazoans generally re- done previously by this group and others in Moore et al. (3) also note that the cobalt sults from defects in digestion, absorption, or elucidating the aerobic biosynthetic pathway (II)intheprecorrin-5A/Band-6A/Bdoes transport mechanisms, although intestinal in- (13, 14), it also reveals previously undescribed not undergo any redox reaction, but as- festation with the fish tapeworm Diphyllo- chemistries that may prove of value in syn- sumes an electron orbital arrangement that bothrium latum, which competes for B12, thetic biology. In addition, knowledge of all is unique for currently characterized biologi- may also result in megaloblastic anemia. Strict steps in both the anaerobic and aerobic co- cal systems. Their data led them to suggest vegans or life-long vegetarians may need to balamin pathways opens possibilities for new, that the metal participates in the midpathway consider B12 supplementation because plants more restricted antibiotic targets, along with reactions, which explains why metallation contain no cobalamin (10). production of cobalamin analogs. must occur before corrin ring decoration. The authors propose that the cobalt contrib- utes by helping to stabilize precorrin-5A/B 1 Hamza I, Dailey HA (2012) One ring to rule them all: Trafficking of 8 Ishida T, et al. (1998) A primitive pathway of porphyrin fi heme and heme synthesis intermediates in the metazoans. Biochim biosynthesis and enzymology in Desulfovibrio vulgaris. Proc Natl and -6A/B. This nding is of interest because Biophys Acta 1823(9):1617–1632. Acad Sci USA 95(9):4853–4858. 9 theanaerobicbiosyntheticpathwaydiffers 2 Girvan HM, Munro AW (2013) Heme sensor proteins. J Biol Chem Gruber K, Puffer B, Kräutler B (2011) Vitamin B12-derivatives- 288(19):13194–13203. enzyme cofactors and ligands of proteins and nucleic acids. Chem from the aerobic pathway in that the cobalt – 3 Moore SJ, et al. (2013) Elucidation of the anaerobic pathway for Soc Rev 40(8):4346 4363. 10 ion is inserted early at the factor II stage, the corrin component of cobalamin (vitamin B ). Proc Natl Acad Sci Pawlak R, Parrott SJ, Raj S, Cullum-Dugan D, Lucus D (2013) How 12 fi which still possesses the porphin ring struc- USA 110:14906–14911. prevalent is vitamin B(12) de ciency among vegetarians? Nutr Rev 71(2):110–117. 4 Graham RM, Deery E, Warren MJ (2009) Vitamin B12: Biosynthesis ture, by CbiX or CbiK (depending upon 11 Raux E, et al. (2003) Identification and functional analysis of of the corrin ring.Tetrapyrroles: Birth, Life and Death, eds Warren MJ, enzymes required for precorrin-2 dehydrogenation and metal ion which enzyme the organism possesses), where- Smith AG (Landes Bioscience, Austin, TX), pp 286–299. insertion in the biosynthesis of sirohaem and cobalamin in Bacillus as in the aerobic pathway cobalt is inserted 5 Warren MJ, Deery E, Rose R-S (2009) Biosynthesis of siroheme megaterium. Biochem J 370(Pt 2):505–516. fi and coenzyme F430. Tetrapyrroles: Birth, Life, and Death, eds after most macrocycle decoration/modi - 12 Proulx KL, Woodard SI, Dailey HA (1993) In situ conversion Warren MJ, Smith AG (Landes Bioscience, Austin, TX), of coproporphyrinogen to heme by murine mitochondria: cation and ring contraction. Metallation – pp 343 351. Terminal steps of the heme biosynthetic pathway. 6 occurs with a,c-diamide hydrogenobryrinic Layer G, Reichelt J, Jahn D, Heinz DW (2010) Structure and Protein Sci 2(7):1092–1098. acid as substrate by CobNST. function of enzymes in heme biosynthesis. Protein Sci 19(6): 13 Warren MJ, Raux E, Schubert HL, Escalante-Semerena JC (2002) 1137–1161. It is worth noting that, with few excep- The biosynthesis of adenosylcobalamin (vitamin B12). Nat Prod Rep 7 Bali S, et al. (2011) Molecular hijacking of siroheme for the 19(4):390–412. tions, organisms that use heme possess the synthesis of heme and d1 heme. Proc Natl Acad Sci USA 108(45): 14 Blanche F, et al. (1993) Parellels and decisive differences in – – entire biosynthetic pathway (1). Whether in 18260 18265. vitamin B12 biosyntheses. Angew Chem Int Ed Engl 32:1651 1653.

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