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MetaCyc Pathway: adenosylcobalamin biosynthesis II (aerobic)
Traceable author statement to experimental support

Pathway diagram: adenosylcobalamin biosynthesis II (aerobic)

If an enzyme name is shown in bold, there is experimental evidence for this enzymatic activity.

Synonyms: adenosylcobalamin biosynthesis II (late cobalt incorporation), vitamin B12 biosynthesis

Superclasses: BiosynthesisCofactors, Prosthetic Groups, Electron Carriers BiosynthesisVitamins BiosynthesisCobalamin BiosynthesisAdenosylcobalamin BiosynthesisDe Novo Adenosylcobalamin Biosynthesis

Some taxa known to possess this pathway include : Brucella melitensis, Pseudomonas denitrificans, Rhodobacter capsulatus, Sinorhizobium meliloti

Expected Taxonomic Range: Archaea, Bacteria

General Background

Adenosylcobalamin (also known as vitamin B12 or coenzyme B12) was discovered in the 1920s after Minot and Murphy reported that they could cure the symptoms of pernicious anaemia by feeding patients with crude liver extract [Minot26] (they received the Nobel prize in 1934 for this discovery). The unknown factor has been isolated and subsequently crystallized in 1948 [Smith48, Rickes48], and was given the name vitamin B12 and, as it was shown to contain a cobalt ion, cobalamin (although this term is used to describe a family of related compounds) [Warren02].

Cobalamins are some of the most structurally complex small molecules made in Nature. They contain a contracted porphinoid ring with a cobalt ion ligated at its center and further held in place by a lower axial base ( 5,6-dimethylbenzimidazole) and a methyl or adenosyl group that function as an upper axial ligand. Their biosynthesis is similarly complex, and requires more than thirty genes. Cobalamin biosynthesis is confined to only some bacteria and archaea [Martens02].

Two main pathways are known for adenosylcobalamin biosynthesis - an aerobic pathway (this pathway) and an anaerobic pathway (see adenosylcobalamin biosynthesis I (anaerobic)). The main differences between these pathways are the timing of the Co2+ ion insertion and the ring-contraction mechanism (for more about ring contraction, see [Rasetti81]). Co2+ is inserted early on in the anaerobic pathway, and rather late in the aerobic pathway. The two different routes then merge at cob(II)yrinate a,c-diamide, and the later part of the pathway is identical, or at least very similar.

About This Pathway

The best characterized aerobic adenosylcobalamin biosynthetic pathway is that of the aerobic bacterium Pseudomonas denitrificans.

The biosynthesis of adenosylcobalamin starts with the biosynthesis of the tetrapyrrole intermediate uroporphyrinogen-III, which is a common intermediate in the biosynthesis of several important compounds, such as heme and chlorophyl. Uroporphyrinogen III is converted to a corrin ring by a complex process that involves (among other things) the attachment of eight methyl groups, all derived from S-adenosyl-L-methionine (SAM). During the process the ring contracts via bonding of carbons C-1 and C-19 to each other, eliminating carbon C-20 from the ring. Both the C-20 carbon and the added C-20 methyl group are lost in the form of acetate.

The intermediates formed prior to the formation of the corrin ring are called precorrins. The precorrins are numbered corresponding to the number of methyl groups that have been already introduced. If two different intermediates have the same number of methyl groups, they are also labeled by A or B (as in precorrin-6A). If there is still some uncertainty about an intermediate, it is given a temporary designation of X or Y.

The first committed step in this pathway is the addition of a methyl group to carbon C-20 of the tetrapyrrole ring to give precorrin-3A. This intermediate, which contains three additional methyl groups (compared with uroporphyrinogen-III), is oxidized by precorrin-3B synthase, using molecular oxygen, to precorrin-3B, an intermediate that has been described as "spring-loaded for contraction" [Warren02]. This intermediate, which has a hydroxy group at carbon C-20, forms a lactone between carbons C-1 and C-2. However, the ring is not contracted until the next enzyme (another methyltransferase) adds a methyl group to carbon C-17. At this point the ring contracts, forming the next intermediate, precorrin-4. Following the addition of four more methyl groups (at positions C-11, C-1, C-5 and C-15) and the release of acetate and CO2, the final precorrin intermediate, precorrin-8x, is formed. This intermediate is the substrate for the enzyme precorrin-8x methylmutase subunit, which finally converts it to a true corrin ring compound, hydrogenobyrinate.

This corrin ring intermediate still requires an extensive modification. Successive amidation reactions transfer two amide groups from two L-glutamine molecules to the carboxy groups a and c, resulting in cob(II)yrinate a,c-diamide, and a Co2+ ion is inserted into the ring by the enzyme cobaltochelatase, forming cob(II)yrinate a,c-diamide - the point where the early-insertion and late-insertion pathways combine.

The Co2+ ion is immediately reduced to Co1+ and is adenosylated to form adenosyl-cobyrinate a,c-diamide. Additional amidation of carboxy groups b, d, e and g generates adenosylcobyrate. At this point the lower ligand base is synthesized and tethered to the corrin ring via a structure known as the nucleotide loop, which is composed of some form of (R)-1-aminopropan-2-ol and 5,6-dimethylbenzimidazole. More information about the biosynthesis of these side chains is provided in the pathways aminopropanol phosphate biosynthesis I and 5,6-dimethylbenzimidazole biosynthesis I (aerobic).

There is some uncertainty about the order in which some of the final reactions of the pathway occur. Depending on the order of the reactions, different intermediates may form. It was initially assumed that (R)-1-aminopropan-2-ol serves as substrate for the enzyme that attaches it to adenosylcobyrate. However, the Km value for (R)-1-aminopropan-2-ol is very high (20mM), suggesting this may not be the natural substrate. Recent findings in Salmonella enterica enterica serovar Typhimurium suggest that the natural substrate is actually (R)-1-amino-2-propanol O-2-phosphate. In this case the product of the reaction is adenosyl-cobinamide phosphate, which is further phosphorylated by GTP to adenosylcobinamide-GDP [Brushaber98]. Even though this has not been proven in Pseudomonas denitrificans, we have decided to present the pathway using this route. Another uncertainty concerns the addition of α-ribazole 5'-phosphate to adenosylcobinamide-GDP. The adenosyl-cobalamin (5'-phosphate) synthase enzyme isolated from Pseudomonas denitrificans accepts both α-ribazole and α-ribazole 5'-phosphate as substrates [Cameron91]. It is not clear whether α-ribazole 5'-phosphate is added directly, generating adenosylcobalamin 5'-phosphate, followed by dephosphorylation to adenosylcobalamin (as shown here), or whether the phosphate group is first removed from α-ribazole 5'-phosphate, in which case α-ribazole is added, and the product is adenosylcobalamin. We have chosen to use the first option in this pathway diagram.

Subpathways: adenosylcobalamin biosynthesis from cobyrinate a,c-diamide I, cob(II)yrinate a,c-diamide biosynthesis II (late cobalt incorporation), tetrapyrrole biosynthesis I (from glutamate), aminopropanol phosphate biosynthesis I, 5,6-dimethylbenzimidazole biosynthesis I (aerobic)

Variants: adenosylcobalamin biosynthesis I (anaerobic)

Created 18-Jan-2001 by Pellegrini-Toole A, Marine Biological Laboratory
Revised 20-Apr-2007 by Caspi R, SRI International
Revised 25-Sep-2013 by Caspi R, SRI International


Brushaber98: Brushaber KR, O'Toole GA, Escalante-Semerena JC (1998). "CobD, a novel enzyme with L-threonine-O-3-phosphate decarboxylase activity, is responsible for the synthesis of (R)-1-amino-2-propanol O-2-phosphate, a proposed new intermediate in cobalamin biosynthesis in Salmonella typhimurium LT2." J Biol Chem 273(5);2684-91. PMID: 9446573

Cameron91: Cameron B, Blanche F, Rouyez MC, Bisch D, Famechon A, Couder M, Cauchois L, Thibaut D, Debussche L, Crouzet J (1991). "Genetic analysis, nucleotide sequence, and products of two Pseudomonas denitrificans cob genes encoding nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase and cobalamin (5'-phosphate) synthase." J Bacteriol 1991;173(19);6066-73. PMID: 1917841

Martens02: Martens JH, Barg H, Warren MJ, Jahn D (2002). "Microbial production of vitamin B12." Appl Microbiol Biotechnol 58(3);275-85. PMID: 11935176

Minot26: Minot, G.R., Murphy, W.P. (1926). "Treatment of pernicious anaemia by a special diet." J. Am. Med. Assoc. 87:470-476.

Rasetti81: Rasetti V, Pfaltz A, Kratky C, Eschenmoser A (1981). "Ring contraction of hydroporphinoid to corrinoid complexes." Proc Natl Acad Sci U S A 78(1);16-19. PMID: 16592942

Rickes48: Rickes, E. L., Brink, N. G., Koniuszy, F. R., Wood, T. R., Folkers, K. (1948). "Crystalline vitamin B12." Science 107: 396-397.

Smith48: Smith, E. L. (1948). "Purification of antipernicious anemia factors from liver." Nature 161: 638-639.

Warren02: Warren MJ, Raux E, Schubert HL, Escalante-Semerena JC (2002). "The biosynthesis of adenosylcobalamin (vitamin B12)." Nat Prod Rep 19(4);390-412. PMID: 12195810

Other References Related to Enzymes, Genes, Subpathways, and Substrates of this Pathway

Alwan89: Alwan AF, Mgbeje BI, Jordan PM (1989). "Purification and properties of uroporphyrinogen III synthase (co-synthase) from an overproducing recombinant strain of Escherichia coli K-12." Biochem J 264(2);397-402. PMID: 2557837

Anderson79: Anderson PM, Desnick RJ (1979). "Purification and properties of delta-aminolevulinate dehydrase from human erythrocytes." J Biol Chem 254(15);6924-30. PMID: 457661

Balg07: Balg C, Blais SP, Bernier S, Huot JL, Couture M, Lapointe J, Chenevert R (2007). "Synthesis of beta-ketophosphonate analogs of glutamyl and glutaminyl adenylate, and selective inhibition of the corresponding bacterial aminoacyl-tRNA synthetases." Bioorg Med Chem 15(1);295-304. PMID: 17049867

Battersby80: Battersby AR, Fookes CJ, Matcham GW, McDonald E (1980). "Biosynthesis of the pigments of life: formation of the macrocycle." Nature 285(5759);17-21. PMID: 6769048

Bernier05: Bernier S, Dubois DY, Habegger-Polomat C, Gagnon LP, Lapointe J, Chenevert R (2005). "Glutamylsulfamoyladenosine and pyroglutamylsulfamoyladenosine are competitive inhibitors of E. coli glutamyl-tRNA synthetase." J Enzyme Inhib Med Chem 20(1);61-7. PMID: 15895686

Blanche89: Blanche F, Debussche L, Thibaut D, Crouzet J, Cameron B (1989). "Purification and characterization of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans." J Bacteriol 171(8);4222-31. PMID: 2546914

Blanche91: Blanche F, Debussche L, Famechon A, Thibaut D, Cameron B, Crouzet J (1991). "A bifunctional protein from Pseudomonas denitrificans carries cobinamide kinase and cobinamide phosphate guanylyltransferase activities." J Bacteriol 1991;173(19);6052-7. PMID: 1655696

Blanche91a: Blanche F, Robin C, Couder M, Faucher D, Cauchois L, Cameron B, Crouzet J (1991). "Purification, characterization, and molecular cloning of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Methanobacterium ivanovii." J Bacteriol 173(15);4637-45. PMID: 1856165

Blanche91b: Blanche F, Couder M, Debussche L, Thibaut D, Cameron B, Crouzet J (1991). "Biosynthesis of vitamin B12: stepwise amidation of carboxyl groups b, d, e, and g of cobyrinic acid a,c-diamide is catalyzed by one enzyme in Pseudomonas denitrificans." J Bacteriol 1991;173(19);6046-51. PMID: 1917839

Blanche92: Blanche F, Maton L, Debussche L, Thibaut D (1992). "Purification and characterization of Cob(II)yrinic acid a,c-diamide reductase from Pseudomonas denitrificans." J Bacteriol 1992;174(22);7452-4. PMID: 1429467

Blanche92a: Blanche F, Thibaut D, Famechon A, Debussche L, Cameron B, Crouzet J (1992). "Precorrin-6x reductase from Pseudomonas denitrificans: purification and characterization of the enzyme and identification of the structural gene." J Bacteriol 1992;174(3);1036-42. PMID: 1732193

Blanche92b: Blanche F, Famechon A, Thibaut D, Debussche L, Cameron B, Crouzet J (1992). "Biosynthesis of vitamin B12 in Pseudomonas denitrificans: the biosynthetic sequence from precorrin-6y to precorrin-8x is catalyzed by the cobL gene product." J Bacteriol 1992;174(3);1050-2. PMID: 1732195

Blanche95: Blanche F., Cameron B., Crouzet J., Debussche L., Thibaut D., Vuilhorgne M., Leeper F. J., Battersby A. R. (1995). "Vitamin B12: how the problem of its biosynthesis was solved." Angewandte Chemie. International edition in English 34(4): 383-411.

Bollivar04: Bollivar DW, Clauson C, Lighthall R, Forbes S, Kokona B, Fairman R, Kundrat L, Jaffe EK (2004). "Rhodobacter capsulatus porphobilinogen synthase, a high activity metal ion independent hexamer." BMC Biochem 5;17. PMID: 15555082

BRENDA14: BRENDA team (2014). Imported from BRENDA version existing on Aug 2014.

Cameron91a: Cameron B, Guilhot C, Blanche F, Cauchois L, Rouyez MC, Rigault S, Levy-Schil S, Crouzet J (1991). "Genetic and sequence analyses of a Pseudomonas denitrificans DNA fragment containing two cob genes." J Bacteriol 173(19);6058-65. PMID: 1917840

Campbell06: Campbell GR, Taga ME, Mistry K, Lloret J, Anderson PJ, Roth JR, Walker GC (2006). "Sinorhizobium meliloti bluB is necessary for production of 5,6-dimethylbenzimidazole, the lower ligand of B12." Proc Natl Acad Sci U S A 103(12);4634-9. PMID: 16537439

Campbell73: Campbell RL, Dekker EE (1973). "Formation of D-1-amino-2-propanol from L-threonine by enzymes from Escherichia coli K-12." Biochem Biophys Res Commun 53(2);432-8. PMID: 4577583

Campbell78: Campbell RL, Swain RR, Dekker EE (1978). "Purification, separation, and characterization of two molecular forms of D-1-amino-2-propanol:NAD+ oxidoreductase activity from extracts of Escherichia coli K-12." J Biol Chem 253(20);7282-8. PMID: 359547

Cantoni84: Cantoni L, Dal Fiume D, Ruggieri R (1984). "Decarboxylation of uroporphyrinogen I and III in 2,3,7,8-tetrachlorodibenzo-p-dioxin induced porphyria in mice." Int J Biochem 16(5);561-5. PMID: 6724109

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Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
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