Vitamin Methyl transfer

Lenhert and Hodgkin (15) revealed with X-ray diffraction techniques that 5 -deoxyadenosylcobalamin (Bi2-coenzyme) contained a cobalt-carbon o-bond (Fig. 3). The discovery of this stable Co—C-tr-bond interested coordination chemists, and the search for methods of synthesizing coen-zyme-Bi2 together with analogous alkyl-cobalt corrinoids from Vitamin B12 was started. In short order the partial chemical synthesis of 5 -de-oxyadenosylcobalamin was worked out in Smith s laboratory (22), and the chemical synthesis of methylcobalamin provided a second B 12-coenzyme which was found to be active in methyl-transfer enzymes (23). A general reaction for the synthesis of alkylcorrinoids is shown in Fig. 4. 

Co corrinoids play central roles in the two classes of enzymic reactions, i.e. methyl transfer mediated by vitamin B,2 and mutase or isomerase reactions catalyzed by coenzyme B. 253 Though there remain many ambiguities, the former is considered to be a combination of Scheme 100, i and its reverse process, and the latter to be represented by Scheme 103. 

Vitamin B]2 is a red crystalline, cobalt-containing compound that can be isolated from the liver. It has a functional role in preventing pernicious anaemia and also serves as a coenzyme in hydrogen and methyl transfer reactions (Co appears to be the only metal in vivo catalyzing C transfer reactions O and N transfers are more common). Vitamin B12 is also a growth-promoting factor for several microorganisms. 

Model reactions have contributed significantly to our understanding of biological processes. Both pyridoxal phosphate (vitamin B6) and Bi2-coenzymes have proved useful in mechanism studies. Methyl transfer reactions to various metals are of environmental significance. In 1968 it was shown that methylcobalamin could transfer a methyl carbanion to mercury(II) salts in aqueous solutions. Recent research on interaction between B12-coenzymes and platinum salts has shown that charged Ptn salts labilize the Co—-C bond. Secondly, the B12-coenzymes are unstable in the presence of platinum salts this observation correlates with the fact that patients who have received cw-platin develop pernicious anemia. 

Vitamin B12 is a biologically active corrinoid, a group of cobalt-containing compounds with macrocyclic pyrrol rings. Vitamin B12 functions as a cofactor for two enzymes, methionine synthase and L-methylmalonyl coenzyme A (CoA) mutase. Methionine synthase requires methylcobalamin for the methyl transfer from methyltetrahydrofolate to homocysteine to form methionine tetrahy-drofolate. L-methylmalonyl-CoA mutase requires adenosylcobalamin to convert L-methylmalonyl-CoA to succinyl-CoA in an isomerization reaction. An inadequate supply of vitamin B12 results in neuropathy, megaloblastic anemia, and gastrointestinal symptoms (Baik and Russell, 1999). 

Wagner et al. (22) isolated Pseudomonas MS which grew on trimethylsul-phonium salts as a sole source of carbon and energy, with the evolution of DMS. A partially purified enzyme preparation catalyzed the transfer of a methyl group from trimethylsulfonium chloride to tetrahydrofolate. Neither S-aaenosyl-methionine nor DMSP functioned in the methyl transfer reaction. The methyltransferase was devoid of vitamin B12, had a molecular weight of about 100,(JOO with a pH optimum of 7.8. 

As a model study of methyl cobalamine (methyl transfer) in living bodies, a methyl radical, generated by the reduction of the /s(dimethylglyoximato)(pyridine)Co3+ complex to its Co1+ complex, reacts on the sulfur atom of thiolester via SH2 to generate an acyl radical and methyl sulfide. The formed methyl radical can be trapped by TEMPO or activated olefins [8-13]. As a radical character of real vitamin B12, the addition of zinc to a mixture of alkyl bromide (5) and dimethyl fumarate in the presence of real vitamin B12 at room temperature provides a C-C bonded product (6), through the initial reduction of Co3+ to Co1+ by zinc, reaction of Co1+ with alkyl bromide to form R-Co bond, its homolytic bond cleavage to form an alkyl radical, and finally the addition of the alkyl radical to diethyl fumarate, as shown in eq. 11.4 [14]. 

In microorganisms, vitamin B12 is involved in a variety of reactions, including methyl transfer the reduction of carbon dioxide to methane, a number... 

Vitamin B12 (cyanocobalamin) 3 is, in fact, not a natural product as the cyanide ligand to the cobalt ion is added during the isolation procedure. Coenzyme B12 (adenosylcobalamin) 4 and methylcobalamin 5 are the true final products of the biosynthetic pathway. Coenzyme 0,2 is the cofactor for a number of enzymic rearrangement reactions, such as that catalysed by methylmalonyl CoA mutase, and methylcobalamin is the cofactor for certain methyl transfer reactions, including the synthesis of methionine. A number of anaerobic bacteria produce related corrinoids in which the dimethylbenzimidazole moiety of the cobalamins (3 - 5) is replaced by other groups which may or may not act as ligands to the cobalt ion, such as adenine orp-cresol [12]. 

Early work in fractionated extracts of both Escherichia coli and liver indicated the participation of a vitamin Bi2-containing protein in the reaction of homocysteine with 5-methyltetrahydrofolate to form methionine and tetrahydrofolate. ° The reaction was stimulated by SAM, although SAM was not the stoichiometric methyl donor. Methylcobalamin was not the primary methyl donor however, it could serve as a methyl donor in the absence of 5-methyltrahydrofolate. It was suggested that methylcobalamin could be a catalytic intermediate in methyl transfer from 5-methyltetrahydrofolate. The role of SAM remained obscure until recent years, when it was found to preserve enzyme activity by maintaining cobalamin as methylcobalamin in the Bi2-protein. 

Stupperich, E. Konle, R. Lehle, M. Corrinoid-dependent Methyl Transfer Reactions in Sporomusa ovata. In Vitamin Biz tid Biz Pfoteins Krautler, B., Arigoni, D., Golding, B. T, Eds. Wiley-VCH Weinheim, 1998 pp 179-187. 

Progress of biological action in enzymatic methyl transfer and rearrangement reactions, medicinal aspects, structure and reactivity, and biosynthesis of vitamin B12 and B 12-coenzymes was very recently comprehensively presented at the 4 European Symposium on Vitamin Bi2 and B 12-Proteins and reviewed in an excellent monograph (70). Therefore it is intended with this contribution to address mainly results from research in the field of vitamin B12 biosynthesis. The procedure makes sense because of two reasons. First, many of the biosynthetic intermediates are closely related to hydroporphyrinoid structures discussed in previous sections and second, reactions of the biosynthetic pathway concern the chemistry of the hydroporphyrinoid and corrinoid frameworks involved, whereas the biochemical reactivity of vitamin B12 is mainly restricted to the central cobalt ion of the corrin macrocycle. 

Inasmuch as TTP, one of the four triphosphate nucleotides needed for DNA synthesis, is formed through the methylation of UMP to TMP which is then further phosphorylated, one may expect that a reduction in methyl transfer would reduce the levels of TTP and thereby cause interference with DNA synthesis and maturation of the red cell. Yet, the pools of TTP in lymphocytes were normal in untreated patients deficient in vitamin Bi2- In contrast, in patients treated with methotrexate a marked drop in the TTP pool is found [166]. A drop in thymidylate synthetase activity in phytohemagglutinin stimulated lymphocytes of patients with pernicious anemia has been described. 

The finding in 1946 by T. Spies and colleagues that thymine can substitute the functions of folic acid and vitamin B12 led to the understanding that both folic acid and vitamin B12 are involved in methyl transfer reactions (Vilter et al. 1950). Donaldson and Keresztesy showed that folic acid can exist in various forms with one-carbon group attached. The coenzyme methylcobalamin was discovered in 1964 (Lindstrand 1964). 

Previously, AdoMet was considered as the sole and universal methyl donor for DNA methylation. Our suggestion of the involvement of CHsCbl in DNA methylation has been confirmed in Micrococcus luteus (Pfohl-Leszkowicz et al., 1991). The authors showed that vitamin B12, CHaCbl and AdoCbl stimulated DNA methylation in cell-free extracts of M. luteus in the presence of rat spleen DNA-methylase and AdoMet at concentrations up to 1 pM at higher concentrations, cobalamins acted as competitive inhibitors of the enzymatic methylation. In addition, the authors found that AdoMet did not inhibit the incorporation of CHs-groups catalyzed by CHsCbl. This observation indicates that AdoMet and CHsCbl act at different sites on the enzyme. In fact, the nucleotide sequence of the cloned mouse DNA methylase has been determined, revealing two domains, one binding AdoMet and another, responsible for methyl transfer (Bestor et al., 1988). 

Photolysis of [Co(CH2R)(L)(Hdmg)2] under oxygen proceeds by insertion of dioxygen into the cobalt carbon bond to provide a solution species for which nmr spectroscopic data is rq)orted. Reduction of this intermediate produces primary alcohols whereas thermolysis produces aldehydes and alcohols. Treatment of [Co oep)] with simple aldehydes and rm-butylhydroperoxide in the presence of sodium borohydride produces cobalt(III) acyls in 65-98% yields. In the absence of the borohydride the yield is reduced. The reaction is proposed to proceed by acyl radical trapping by the Co(n) centre. Methyl transfer in a protein free model of vitamin B12 dependent methyl transf enzymes has been studied. These systems convert homocysteine to methionine in nature. Trimethyl-phenylammonium icm reacts with the CoG) centre in cobalamin producing methylcobalamin. ... 

FIGURE 18.29 Vitamin B19 functions as a coenzyme in intramolecular rearrangements, reduction of ribonucleotides, and methyl group transfers. 

Methyl-tetrahydro folic acid is furthermore, together with vitamin B12 and B6, required to regenerate homocysteine (see Vitamin B12, Fig. 1). Homocysteine results when methionine is used as a substrate for methyl group transfer. During the last few years, homocysteine has been acknowledged as an independent risk factor in atherosclerosis etiology. Folic acid supplementation can help reduce elevated homocysteine plasma levels and is therefore supposed to reduce the risk of atherosclerosis as well [2]. 

Compared with other vitamins, the chemical structures of both folic acid and B12 are complex. They are prosthetic groups for the enzymes that catalyse the transfer of the methyl group (-CH3) between compounds (one-carbon metabolism). The -CH3 group is chemically unreactive, so that the chemistry for the transfers is difficult, requiring complex structures for catalysis. 

The functions of folic acid and vitamin B12 are very closely linked, especially in what is known as one carbon metabolism or methyl group transfer. 

Small methyl groups are important in the stractnre of some small compounds, nucleotides, some bases in DNA mole-cnles and in postranslational modification of amino acids in proteins. The transfer of a single carbon atom is important in the synthesis of purine nncleotides. The componnds involved in the whole process of methyl gronp transfer, and are carbon metolism, are methionine, homocysteine, serine and the vitamins, folic acid and B12. 

Two essential enzymatic reactions in humans require vitamin B12 (Figure 33-2). In one, methylcobalamin serves as an intermediate in the transfer of a methyl group from /V5-methyltetrahydrofolate to homocysteine, forming methionine (Figure 33-2A Figure 33-3, section 1). Without vitamin B12, conversion of the major dietary and storage folate, N5-... 

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