Cobalamin biosynthesis


Cobalamin biosynthesis is the process by which bacteria and archea make cobalamin, vitamin B12. Many steps are involved in converting aminolevulinic acid via uroporphyrinogen III and adenosylcobyric acid to the final forms in which it is used by enzymes in both the producing organisms and other species, including humans who acquire it through their diet.

Cobalamin

Cobalamin is the largest and most structurally complex vitamin. It consists of a modified tetrapyrrole, a corrin, with a centrally chelated cobalt and is usually found in one of two biologically active forms: methylcobalamin and adenosylcobalamin. Most prokaryotes, as well as animals, have cobalamin-dependent enzymes that use it as a cofactor, whereas plants and fungi do not use it. In bacteria and archaea, these enzymes include methionine synthase, ribonucleotide reductase, glutamate and methylmalonyl-CoA mutases, ethanolamine ammonia-lyase, and diol dehydratase. In certain mammals, cobalamin is obtained through the diet, and is required for methionine synthase and methylmalonyl-CoA mutase. In humans, it plays essential roles in folate metabolism and in the synthesis of the citric acid cycle intermediate, succinyl-CoA.

Overview of cobalamin biosynthesis

There are at least two distinct cobalamin biosynthetic pathways in bacteria:
Either pathway can be divided into two parts:
A further type of synthesis occurs through a salvage pathway, where outside corrinoids are absorbed to make B12.
Species from the following genera and the following individual species are known to synthesize cobalamin: Propionibacterium shermanii, Pseudomonas denitrificans, Streptomyces griseus, Acetobacterium, Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus, Clostridium, Corynebacterium, Flavobacterium, Lactobacillus, Micromonospora, Mycobacterium, Nocardia, Proteus,
Rhizobium, Salmonella, Serratia, Streptococcus and Xanthomonas.

Detail of steps up to formation of uroporphyrinogen III

In the early steps of the biosynthesis, a tetrapyrrolic structural framework is created by the enzymes deaminase and cosynthetase which transform aminolevulinic acid via porphobilinogen and hydroxymethylbilane to uroporphyrinogen III. The latter is the first macrocyclic intermediate common to haem, chlorophyll, sirohaem and cobalamin itself.

Detail of steps from uroporphyrinogen III to cob(II)yrinic acid a,c-diamide in aerobic organisms

The biosynthesis of cobalamin diverges from that of haem and chlorophyll at uroporphrinogen III: its transformation involves the sequential addition of methyl groups to give intermediates that were given trivial names according to the number of these groups that have been incorporated. Hence, the first intermediate is precorrin-1, the next is precorrin-2 and so on. The incorporation of all eight additional methyl groups which occur in cobyric acid was investigated using 13C methyl-labelled S-adenosyl methionine. It was not until scientists at Rhône-Poulenc Rorer used a genetically-engineered strain of Pseudomonas denitrificans, in which eight of the cob genes involved in the biosynthesis of the vitamin had been overexpressed, that the complete sequence of methylation and other steps could be determined, thus fully establishing all the intermediates in the pathway.

From uroporphyrinogen III to precorrin-2

The enzyme CobA catalyses the two chemical reactions
reactions

From precorrin-2 to precorrin-3A

The enzyme CobI catalyzes the reaction

From precorrin-3A to precorrin-3B

The enzyme CobG catalyzes the reaction
This enzyme is an oxidoreductase that requires oxygen and hence the reaction can only operate under aerobic conditions. The naming of these precorrins as 3A and 3B reflects the fact that each contains three more methyl groups than uroporphyrinogen III but with different structures: in particular, precorrin-3B has an internal γ-lactone ring formed from the ring A acetic acid sidechain closing back on to the macrocycle.

From precorrin-3B to precorrin-4

The enzyme CobJ continues the theme of methyl group insertion by catalysing the reaction
Importantly, during this step the macrocycle ring-contracts so that the product contains for the first time the corrin core which characterises cobalamin.

From precorrin-4 to precorrin-5

Methyl group insertions continue when the enzyme CobM catalyses the reaction
The newly-inserted methyl group is added to ring C at the carbon attached to the methylene bridge to ring B. This is not its final location on cobalamin as a later step involves its rearrangement to an adjacent ring carbon.

From precorrin-5 to precorrin-6A

The enzyme CobF catalyzes the reaction
This conversion removes the acetyl group located at position 1 of the ring system in precorrin-4 and replaces it with a newly-introduced methyl group. The name of the product, precorrin-6A, reflects the fact that six methyl groups in total have been added to uroporphyrinogen III up to this point. However, since one of these has been extruded with the acetate group, the structure of precorrin-6A contains just the remaining five.

From precorrin-6A to precorrin-6B

The enzyme CobK now reduces a double bond in ring D by catalysing the reaction
Precorrin-6B therefore differs in structure from precorrin-6A only by having an extra two hydrogen atoms.

From precorrin-6B to precorrin-8

The enzyme CobL has two active sites, one catalysing two methyl group additions and the other the decarboxylation of the CH2COOH group on ring D, so that this substituent becomes a simple methyl group

From precorrin-8 to hydrogenobyrinic acid

The enzyme CobH catalyzes a rearrangement reaction
The result is that the methyl group that had been added to ring C is isomerised to its final location, an example of intramolecular transfer.

From hydrogenobyrinic acid to hydrogenobyrinic acid a,c-diamide

The next enzyme in the pathway, CobB, converts two of the eight carboxylic acid groups into their primary amides in the reaction

From hydrogenobyrinic acid a,c-diamide to cob(II)yrinic acid a,c-diamide

insertion into the macrocycle is catalysed by the enzyme Cobalt chelatase in the reaction
It is at this stage that the aerobic pathway and the anaerobic pathway merge, with later steps being chemically identical.

Detail of steps from uroporphyrinogen III to cob(II)yrinic acid a,c-diamide in anaerobic organisms

Many of the steps beyond uroporphyrinogen III in anaerobic organisms such as Bacillus megaterium involve chemically similar but genetically distinct transformations to those in the aerobic pathway.

From precorrin-2 to cobalt-sirohydrochlorin

The key difference in the pathways is that cobalt is inserted early in anaerobic organisms by first oxidising precorrin-2 to its fully aromatised form sirohydrochlorin and then to that compound's cobalt complex. The reactions are catalysed by CysG and Sirohydrochlorin cobaltochelatase.

From cobalt-sirohydrochlorin to cobalt-factor III

As in the aerobic pathway, the third methyl group is introduced by a methyltransferase enzyme, CbiL in the reaction

From cobalt-factor III to cobalt-precorrin-4

Methylation and ring contraction to form the corrin macrocycle occurs next, catalysed by the enzyme Cobalt-factor III methyltransferase
In this pathway, the resulting material has contains a δ-lactone, a six-membered ring, rather than the γ-lactone of precorrin-3B.

From cobalt-precorrin-4 to cobalt-precorrin-5A

The introduction of the methyl group at C-11 in the next step is catalysed by Cobalt-precorrin-4 methyltransferase in the reaction

From cobalt-precorrin-5A to cobalt-precorrin-5B

The scene is now set for the extrusion of the two-carbon fragment corresponding to the acetate released in the fomation of precorrin-6A in the aerobic pathway. In this case the fragment released is acetaldehyde and this is catalysed by CbiG in the reaction

From cobalt-precorrin-5B to cob(II)yrinic acid a,c-diamide

The steps from cobalt-precorrin-5B to cobyrinic acid a,c-diamide in the anaerobic pathway are essentially chemically identical to those in the aerobic sequence. The intermediates are called cobalt-precorrin-6A, cobalt-precorrin-6B, cobalt-precorrin-8 and cobyrinic acid and the enzymes / reactions involved are Cobalt-precorrin-5B -methyltransferase ; Cobalt-precorrin-6A reductase ; Cobalt-precorrin-7 -methyltransferase , Cobalt-precorrin-8 methylmutase and CbiA /. The final enzyme forms cobyrinic acid a,c-diamide as the two pathways converge.

Detail of steps from cob(II)yrinic acid a,c-diamide to adenosylcobalamin

Aerobic and anaerobic organisms share the same chemical pathway beyond cobyrinic acid a,c-diamide and this is illustrated for the cob gene products.

From cob(II)yrinic acid a,c-diamide to adenosylcobyric acid

The cobalt is reduced to cobalt by the enzyme Cobyrinic acid a,c-diamide reductase and then the enzyme Cobyrinic acid a,c-diamide adenosyltransferase attaches an adenosyl ligand to the metal in reaction. Next, the enzyme CobQ converts all the carboxylic acids, except the propionic acid on ring D, to their primary amides.

From adenosylcobyric acid to adenosylcobinamide phosphate

In aerobic organisms, the enzyme CobCD now attaches -1-amino-2-propanol to the propionic acid, forming adenosylcobinamide and the enzyme CobU phosphorylates the terminal hydroxy group to form adenosylcobinamide phosphate. The same final product is formed in anaerobic organisms by direct reaction of adenosylcobyric acid with -1-amino-2-propanol O-2-phosphate catalysed by the enzyme CbiB.

From adenosylcobinamide phosphate to adenosylcobalamin

In a separate branch of the pathway, 5,6-dimethylbenzimidazole is biosynthesised from flavin mononucleotide by the enzyme 5,6-dimethylbenzimidazole synthase and converted by CobT in reaction to alpha-ribazole 5' phosphate. Then the enzyme CobU activates adenosylcobinamide phosphate by formation of adenosylcobinamide-GDP and CobV links the two substrates to form Adenosylcobalamin-5'-phosphate.
In the final step to the coenzyme, CobC removes the 5' phosphate group in the reaction
The complete biosynthetic route involves a long linear path that requires about 25 contributing enzyme steps.

Other pathways of cobalamin metabolism

Salvage pathways in prokaryotes

Many prokaryotic species cannot biosynthesize adenosylcobalamin, but can make it from cobalamin. These organisms are capable of cobalamin transport into the cell and its conversion to the required coenzyme form. Even organisms such as Salmonella typhimurium that can make cobalamin also assimilate it from external sources when available. Uptake into cells is facilitated by ABC transporters which absorb the cobalamin through the cell membrane.

Cobalamin metabolism in humans

In humans, dietary sources of cobalamin are bound after ingestion as transcobalamins. They are then conveted to the coenzyme forms in which they are used. Methylmalonic aciduria and homocystinuria type C protein is the enzyme which catalyzes the decyanation of cyanocobalamin as well as the dealkylation of alkylcobalamins including methylcobalamin and adenosylcobalamin.