Illuminating the black box of B₁₂ biosynthesis

Tetrapyrroles are nearly ubiquitous in nature as participants in a wide variety of biological reactions that are central to life, such as electron transfer, gas binding, and one-carbon metabolism (1, 2). Because of their diverse colors—for example the greens of plant chlorophylls, reds of blood, and blues and browns of avian eggs—tetrapyrroles have been called the “pigments of life.” The chemical diversity of cyclic tetrapyrroles owes much to their ability to coordinate a variety of redox active metals in a fashion that allows for fine-tuning of midpoint potentials, increasing or decreasing reactivity of the metal center, and providing protection against undesirable side reactions. One finds magnesium in chlorophyll, iron in hemes, nickel in factor F₄₃₀, and cobalt in cyano-cobalamin (vitamin B₁₂) (Fig. 1). Although hemes and chlorophyls are synthesized by both prokaryotes and eukaryotes, factor F₄₃₀, which is involved in methanogenesis, is produced only by some archae, and cobalamin synthesis is found only in bacteria and archae. With regard to cobalamin, it has long been known that two distinct pathways exist for its biosynthesis, one aerobic and the other anaerobic. Whereas the genes required for both aerobic and anaerobic synthesis have been known, the actual mechanism for synthesis via the anaerobic pathway has remained a large “black box” in what is one of the longest known biosynthetic pathways. However, the contents of this box have now been identified and characterized, thanks to an elegant study that can properly be called a tour de force by Moore et al. at the University of Kent (3).

Fig. 1.

The family of tetrapyrroles originating from uroporphyrinogen III. The central metal atom is colored to represent the color of the metallo-tetrapyrrole.

Research into the metabolism of metallo-tetrapyrroles and the biological and medical impact of disordered synthesis or degradation of these compounds has been ongoing for the past century, so that we now have a reasonable understanding of this process (1), with 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 either succinyl CoA and glycine in metazoans, or glutamyl tRNA in most other organisms (6). The next three enzymes, porphobilinogen synthase, hydroxymethylbilane 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 biological importance in methanogenesis, factor F₄₃₀’s biosynthetic pathway has yet to be elucidated, although it shares the common pathway to uroporphyrinogen III. Given that this compound, which is a tetrahydroporphyrinogen, is the most reduced of those previously mentioned and the only known metallotetrapyrrole with nickel as the chelated metal, its biosynthesis can be anticipated to possess unique features (5). Cobalamin synthesis also branches off at uroporphyrinogen III, but here one of the early steps is the condensation of the porphin ring structure into a corrin by elimination of one of the bridging methine carbons (4).

Moore et al. tackled illumination of the anaerobic cobalamin synthesis black box by taking advantage of the ability to express at high levels and purify all 14 pathway enzymes in soluble forms from Bacillus megaterium (3). This process allowed them to not only characterize each step of the black box, but also to synthesize in good yield the end product, cobyrinic acid, from the starting compound 5-aminolevulinate, by using a mixture of all 14 enzymes. The integration of UV-Vis spectroscopy, LC-MS, and electron paramagnetic resonance allowed for the identification of all intermediates and revealed some unexpected and exciting findings about this long and complex biosynthetic pathway. These investigators determined that the intermediate precorrin 5B is the oxygen-sensitive intermediate in the pathway. This observation is a major accomplishment that answers a long-standing question as to why this route to cobalamin is oxygen-sensitive. Although there were previous studies hinting at what may have been going on in the black box, the current data now provide compelling evidence and clearly settle the question.

Moore et al. (3) also note that the cobalt (II) in the precorrin-5A/B and -6A/B does not undergo any redox reaction, but assumes an electron orbital arrangement that is unique for currently characterized biological systems. Their data led them to suggest that the metal participates in the midpathway reactions, which explains why metallation must occur before corrin ring decoration. The authors propose that the cobalt contributes by helping to stabilize precorrin-5A/B and -6A/B. This finding is of interest because the anaerobic biosynthetic pathway differs from the aerobic pathway in that the cobalt ion is inserted early at the factor II stage, which still possesses the porphin ring structure, by CbiX or CbiK (depending upon which enzyme the organism possesses), whereas in the aerobic pathway cobalt is inserted after most macrocycle decoration/modification and ring contraction. Metallation occurs with a,c-diamide hydrogenobryrinic acid as substrate by CobNST.

It is worth noting that, with few exceptions, organisms that use heme possess the entire biosynthetic pathway (1). Whether in the Metazoa or prokaryotes, dietary heme is generally broken down and the iron released for reutilization, frequently to make heme. Similarly, chlorophylls and factor F₄₃₀ are synthesized by the organisms that possess them and are not acquired from dietary sources. This process is different for cobalamin because it is synthesized only in prokaryotes; metazoans—which require cobalamin—cannot synthesize it and, therefore, must acquire it from their diet.

Cobalamins are essential for three enzyme-catalyzed reactions: methyltransferases, isomerases, and reductive dehalogenases (9). In humans two enzymes require cobalamin: methylmalonyl CoA mutase, which converts methylmalonyl CoA into succinyl CoA, and 5-methyltetrahydrofolate-homocysteine methyltransferase (methionine synthase), which is required for recycling of tetrahydrofolate via the synthesis of methionine from homocysteine. Insufficient cobalamin can result in nerve myelin damage from accumulation of methylmalonyl CoA, and megaloblastic anemia because of an inability to regenerate tetrahydrofolate for thymine synthesis. Dietary folate supplementation can ameliorate the anemia, but not myelin damage or accumulation of homocysteine (homocysteinuria), which may contribute to stroke and cardiac infarction. Because most nonvegetarian diets provide a reasonable source of B₁₂, deficiency of cobalamin in metazoans generally results from defects in digestion, absorption, or transport mechanisms, although intestinal infestation with the fish tapeworm Diphyllobothrium latum, which competes for B₁₂, may also result in megaloblastic anemia. Strict vegans or life-long vegetarians may need to consider B₁₂ supplementation because plants contain no cobalamin (10).

In addition to identifying black box reactions in the anaerobic biosynthesis of cobalamin, the work of Moore et al. (3) provides observations with a potentially much broader impact. First is the characterization of a nonredox role for cobalt in the reaction. The putative role cobalt plays has not been described previously in a biological system, but the current observation may prompt researchers to reexamine other cobalt-containing enzymes. Second is the observation that a mixture containing all pathway enzymes functions more effectively than individual enzymes to produce product. This finding is not unlike what has been reported for siroheme biosynthesis (7, 11) and hinted at for protoheme synthesis (12). Given the reactivity of tetrapyrrole biosynthetic pathway intermediates, it is not a surprise that evolution has designed pathway enzymes to hold on to products until downstream acceptors are available. Such an approach, either alone or with substrate channeling, may represent a generic approach to minimizing potential toxic cellular effects of free pathway intermediates or nonproductive loss of reactive intermediates: a clear example of how Nature may have evolved complex pathways with reactive intermediates.

Although the current contribution helps bring some closure to the B₁₂ biosynthesis field with the illumination of the anaerobic black box components, to complement work done previously by this group and others in elucidating the aerobic biosynthetic pathway (13, 14), it also reveals previously undescribed chemistries that may prove of value in synthetic biology. In addition, knowledge of all steps in both the anaerobic and aerobic cobalamin pathways opens possibilities for new, more restricted antibiotic targets, along with production of cobalamin analogs.