B₁₂ (Cobalamin(  

Cobalamins are complex organometallic cofactors containing a central cobalt atom that is coordinated equatorially to four nitrogen atoms provided by the corrin ring. The two cofactor forms of this vitamin are adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl). A variety of other forms have been described in which the upper ligand is a water, glutathione, or cyano group, but the physiological significance of these forms is unknown. In nature, this cofactor is found in association with two enzyme subfamilies, the AdoCbl-dependent isomerases that catalyze 1,2 rearrangement reactions and the MeCbl-dependent methyltransferases that catalyze transmethylation reactions. Members of both subfamilies are fairly prevalent in the bacterial world, where the isomerases are involved in fermentative pathways, and the methyltransferases are involved in pathways leading to methionine, acetate, or methane. In mammals, only two B₁₂-dependent enzymes are known, the cytoplasmic methionine synthase and the mitochondrial methylmalonyl-CoA mutase. In this review, the molecular biological aspects of B₁₂ biosynthesis, enzymology and diseases will be discussed briefly.

1. B₁₂ Biosynthesis

 The structural cousins, vitamin B₁₂, heme, and chlorophyll, are constructed from a common template that is derived from the precursor, 5-aminolevulinic acid. Methylation at C2 converts the common intermediate, uroporphyrinogen III, to precorrin-1 and commits it to B₁₂ biosynthesis. Thereafter, a biosynthetic assembly line, which includes a series of methylations, ring contraction (between rings A and D), cobalt insertion, alkylation, amidations, and nucleotide loop assembly reactions, takes the cofactor to its final form. Two separate pathways lead to corrin ring biosynthesis in the aerobic and anaerobic worlds. They run parallel at some steps and diverge at others. A unique structural feature of B₁₂ is the presence of a nucleotide loop that terminates in dimethylbenzimidazole, which can serve as the lower axial ligand to cobalt. The pathways leading to the assembly of dimethylbenzimidaole in aerobic and anaerobic organisms are distinct, but their details remain to be elucidated (5). The genes encoding corrin biosynthesis functions have been cloned from Pseudomonas denitrificans, where they are organized in clusters scattered along the genome  and from Salmonella typhimurium, where they are clustered at 41 min. The transcription factor PocR regulates the transcription of the cobalamin biosynthetic and propanediol utilization (dependent on a B12 enzyme) genes in S. typhimurium (6, 7).

2. B₁₂ Enzymes

The cofactor role of AdoCbl was first described by Barker and co-workers in glutamate mutase (8), followed a few years later by the discovery of MeCbl in methionine synthase (9). B₁₂-dependent enzymes catalyze chemically difficult reactions and manipulate the reactive cobalt–carbon bond in radically different ways. In the methyltransferase subfamily, cobalamin is the initial acceptor of the methyl group donated from substrates such as methyltetrahydrofolate and methanol, and alkyltransfer to and from the cobalamin occurs via heterolytic cleavage of the cobalt–carbon bond. In the isomerases, the cobalt–carbon bond is broken homolytically, as the enzymes catalyze 1,2 rearrangement reactions. The migration of a diversity of groups, ranging from carbon (in methylmalonyl-CoA mutase, glutamate mutase, and methylene glutarate mutase) to nitrogen (in ethanolamine ammonia lyase and b-lysine mutase) and oxygen (in diol dehydrase), is catalyzed by the isomerases. The genes encoding a number of methyltransferases and isomerases have been cloned and the structures of the B₁₂-binding domain of the Escherichia coli methionine synthase (11) and of the Propionibacterium shermanii methylmalonyl-CoA mutase (12) have been determined. In both enzymes, the cofactor is bound in an extended conformation in which the intramolecular base, dimethylbenzimidazole, is removed from the cobalt and replaced by a histidine residue donated by the protein. Another important member of the B₁₂ family of enzymes is ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides (13). The reaction mechanisms of B₁₂-dependent enzymes are discussed in a number of reviews (10, 14-18).

3. B₁₂-Related Diseases

 Functional B₁₂ deficiency can result from either nutritional insufficiency or from genetic defects (19, 20) It is manifested clinically by a combination of symptoms including hematological and neurological abnormalities, methylmalonic aciduria and homocystinuria, depending on whether one or both B₁₂-dependent enzymes is affected. The genetic defects or inborn errors of cobalamin metabolism can result from impairments in uptake, transport, or enzymatic function and are inherited as autosomal recessive traits. Cobalamin absorption and transport abnormalities resulting from impairments in intrinsic factor, its receptor, or in transcobalamin (TC) II have been described. The cDNA encoding human intrinsic factor (21) and TC II (22) have been cloned and mapped, and genetic defects in TC-II in patient cell lines have been identified (23).

Intracellular cobalamin metabolism is complex, compartmentalized, and dependent on several enzymes. Defects in the early steps following internalization of TC II/cobalamin affect both MeCbl and AdoCbl syntheses and fall into the cbl C, D, and F genetic complementation groups. The identities of the proteins encoded by these loci and their precise functions are unknown. MeCbl synthesis is specifically compromised in cblG and cblE patients. Cloning of the cDNA encoding methionine synthase (24-26) and identification of mutations correlated with the cblG phenotype (25,( 27indicate that this locus represents methionine synthase. AdoCbl synthesis is specifically impaired in cblA, B, and mut patients. The defect in cblB and mut patients is in cobalamin adenosyltransferase and methylmalonyl-CoA mutase, respectively. The cDNA encoding methylmalonyl-CoA mutase has been cloned (28), and a number of pathogenic mutations have been described (29).

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