Bi2-dependent Methyltransferases

FIGURE 15.10 (a) The three components involved in the Bi2-dependent methyltransferases. (b) TheMT2 enzymes have a thiol group which 

FIGURE 15.11 Reactions catalysed by cobalamin-dependent methionine synthase. (From Banerjee Ragsdale, 2003. Reprinted with permission from Annual Reviews.) 

As pointed out earlier, a third class of B 12-dependent enzymes, present in anaerobic microbes, carry out reductive dehalogenation reactions, which play an important role in the detoxification of chlorinated aliphatic and aromatic compounds, among which are many important man-made pollutants (El Fantroussi et al., 1998). The role of Bi2 in this class of enzymes is not clear — possibly by formation of an organocobalt adduct, as in the case of methyltransferases or alternatively by the corrinoid serving as an electron donor. 

FIGURE 15.12 The modular structure of methionine synthase. The four domains are connected by flexible hinges which allow the CHstetrahydrofolate-, AdoMet-, or homocystein-binding domains to alternatively access the Bi2-binding domain. From Banerjee Ragsdale, 2003. Reprinted with permission from Annual Reviews.) 

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The Bi2-dependent methyltransferases play an important role in amino acid metaboHsm in many organisms (including humans) as well as in one-carbon... 

The investigations of Woods and his collaborators (1965) on methionine biosynthesis in E. coli first brought to light the fact that this organism possesses an alternate route for de novo methyl group formation. The enzymes involved are the same as those concerned in the Bi2-dependent pathway until the terminal methyl transfer step. Escherichia coli possesses a second, non-Bi2 methyltransferase which is physically separable from the Bi2-dependent methyltransferase. The non-Bi2-enzyme has recently been purified to near homogeneity from extracts of a derepressed methionine auxotroph and obtained in crystalline form. It represents nearly 5% of the soluble protein in derepressed cells and 3% in wild-type cells (Whitfield et al, 1970). The requirements of the enzyme for activity contrast sharply with those of the Bi2-dependent methyltrans-... 

Methylation is the addition of a carbon atom to a molecule, usually causing a change in the function of the methylated molecule. For example, methylation of the neurotransmitter dopamine by catechol-O-methyltransferase renders it inactive. With only two exceptions, 5-adenosylmethionine (SAM), an activated form of the essential amino acid methionine, is the methyl donor for each of the more than 150 methylation reactions, which regulate a large number of cellular functions. One exception is methylation of homocysteine (HCY) to methionine by the cobalamin (vitamin Bi2)-dependent enzyme methionine synthase, which utilizes 5-methyltetrahydrofolate (methylfolate) as the methyl donor, serving to complete the methionine cycle of methylation, as illustrated in Fig. 1 (lower right). Notably, HCY formation from S-adenosylhomocysteine (SAH) is reversible and, as a result, any decrease in methionine synthase activity will be reflected as an increase in both HCY and SAH. This is significant because SAH interferes with SAM-dependent methylation reactions, and a decrease in methionine synthase activity will decrease all of these reactions. Clearly methionine synthase exerts a powerful influence over cell function via its control over methylation. 

The methylation also required a small amount of S-adenosylmethionine (AdoMet) its competitive inhibitor, adenosylhomocysteine, when added to the incubation mixture in the presence of CHaCbl, blocked the transfer of methyl groups to DNA. Furthermore, it appears that methylases of Bn and Bi2-deficient cells might use different donors of CHs-groups. The methylase of vitamin Bi 2-deficient cells showed a higher affinity for AdoMet and catalyzed efficiently the methylation of both cytosine and adenine. The methylase of B " cells methylated adenine, but not cytosine with AdoMet. It is possible that AdoMet is used in the absence of CHaCbl as the natural donor of CH3 groups for DNA methylation. In the presence of CHsCbl, additional methylation is specifically catalyzed by a vitamin B -dependent methyltransferase. It seems likely that either there are two separate methylases or one enzyme with two coenzyme sites, one binding AdoMet, and another CHsCbl... 

In contrast to coenzyme Bi2, where the alkyl moiety serves purely in a catalytic role, the alkyl group of methyl cobamides (MeCba s) is utilized as a reagent by MeCba-dependent enzymes it is only the cobamide portion of the coenzyme that is catalytic. The cobamide-dependent methyl transferases have been reviewed [11,24-27,165], Three cobamide-dependent methyl transferases have been studied in some cases, more than one protein is required. The Bi2 proteins include methionine synthase (officially called 5-methyltetrahydrofolate-L-homocysteine-S-methyltransferase [HCM] EC 2.1.1.13) MeCba-dependent enzyme from Meth-anosarcina barkeri (MT 0 and the corrinoid/Fe-S protein from Clostridium ther-moaceticum. 

Because of the complexity of the enzymatic systems involved in coenzyme Bi2 chemistry there are several reports on the purification of B 12-dependent enzymes or B 12-binding proteins by vitamin B12 affinity adsorbents. In fact, for purification of enzymes or proteins, affinity chomatography has been widely used as one of the most attractive methods (270). For that purpose, the synthesis of a cobalamin-Sepharose insoluble support has been prepared and applied to the purification of iV -methyltetrahydrofolate-homocysteine cobalamin methyltransferase from E. coll The scheme for the synthesis of the solid support is summarized in Fig. 6.14. 

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