N-methyl tetrahydrofolate

Methionine synthase is a vitamin B 12-dependent enzyme that needs N -methyl tetrahydrofolate (THF) as a coenzyme (Fig. 47.1). It catalyses the transfer of the methyl group from A/ -methyl THF to homocysteine to form methionine. When methionine synthase activity is deficient homocysteine accumulates, causing hyperhomocysti-... 

Methylation of homocysteine to methionine can be accomplished by one of several sequences. A major route is from a N -methyl-tetrahydrofolic acid (CHg—FH4) derivative. In some organisms a coenzyme derivative of vitamin B12 is also required, where it functions in its reduced form (B12, in the following scheme) as an intermediate methyl carrier ... 

The other major class of antimalarials are the folate synthesis antagonists. There is a considerable difference in the drug sensitivity and affinity of dihydrofolate reductase enzyme (DHFR) between humans and the Plasmodium parasite. The parasite can therefore be eliminated successfully without excessive toxic effects to the human host. DHFR inhibitors block the reaction that transforms deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) at the end of the pyrimidine-synthetic pathway. This reaction, a methylation, requires N °-methylene-tetrahydrofolate as a carbon carrier, which is oxidized to dihydrofolate. If the dihydrofolate cannot then be reduced back to tetrahydrofolate (THF), this essential step in DNA synthesis will come to a standstill. 

There is at present no unifying concept which would explain the presence of optically active TIQs, TIQ-1-carboxylic acids, and noralkaloids in mammalian systems. The conclusion reached by several investigators that methylenetetrahydrofolate is responsible for the N-methylation of phenethylamines and indolylethylamines (221,222) makes it likely that the one-carbon unit present at C-1 in the noralkaloids is derived from formaldehyde, formed via nonenzymatic disassociation of methylene-tetrahydrofolates (223,224). 

Tetrahydrofolate accepts methyl groups, usually from serine. The product, N, N °-methylene-tetrahydrofolate, is the central compound in 1-carbon metabolism. Tetrahydrofolate can also accept a methyl group from the complete breakdown of glycine. In Figure 5-2, only the N, C , and atoms of the pteroic acid are shown for clarity. 

The donation of the methyl group from N, N -methylene tetrahydrofolate leads to the oxidation of the cofactor to dihydrofolate. This points to the importance of dihydrofolate reductase (DHFR) in the functioning of thymidylate synthase. Thus, synthesis of TMP requires a supply of both methyl groups—for example, from serine— and reducing equivalents. 

The key intermediate in the catalytic pathway is the supemucleophile cob(l)alamin, which attacks A -methyl-tetrahydrofolate, generating tetrahydrofolate and MeCbl. Then homocysteine (probably as its thiolate) attacks MeCbl, which yields methionine and regenerates cob(l)alamin (Scheme 2). The demethylation of A -methyltetrahydrofolate is not trivial, even for the supemucleophilic cob(l)alamin, and considerable efforts have been invested into understanding this reaction, dubbed improbable by Duilio Arigoni. The obvious mode of activation is by proton transfer to N-5 of A -methyl tetrahydrofolate, but as this is weakly basic (pAa 5.1) the nature of the proton source and mode of transfer has been difficult to pin down. Recent research from the Matthews group has shown how the reactivities of cob(I)alamin and methylcobalamin are modulated by the ligand trans to the lone pair of cob(l)alamin and methyl group of methylcobalamin (21). 

It is interesting that E. coli contains two genes that code for methionine synthase metH for the cobalamin-dependent enzyme and metE for a cobalamin-independent enzyme that depends on an active site Zn + to stabilize deprotonated homocysteine (24). This thiolate species demethylates A -methyl-tetrahydrofolate, which is activated by proton transfer to N-5. MetE is less active ( 100 x ) than MetH, and so in the absence of Bi2 E. coli it produces much more MetE to compensate for the lack of MetH. 

Methyl trap The sequestering of tetrahydrofolate as N -methyl THF because of decreased conversion of homocysteine to methionine as a result of a deficiency of methionine synthase or its cofactor, cobalamin (vitamin B ). 

Figure 4-2. Structures of the various 1-carbon carriers of tetrahydrofolate (THF). THF can carry one-carbon units in the oxidation states of methanol (N -methyl THF), formaldehyde (N, N °-methylene THF) or formic acid (remaining structures).

Decreased methylation of deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP), a reaction that requires N, N °-methyl-ene tetrahydrofolate as a coenzyme (see Fig. 40.5), leads to an increase in the intracellular dUTP/dTTP ratio. This change causes a significant increase in the incorporation of uracil into DNA. Although much of this uracil can be removed by DNA repair enzymes, the lack of available dTTP blocks the step of DNA repair catalyzed by DNA polymerase. The result is fragmentation of DNA as well as blockade of normal DNA rephcation. 

Cells that are synthesizing DNA mnst also be able to synthesize deoxy thymidine triphosphate (dTTP). The key step in the synthesis is the conversion of dUMP to dTMP via thymidylate synthetase. The reaction reqnires a sonrce of N, -methylene tetrahydrofolate to provide the methyl gronp. In this reaction the tetrahydrofolate is oxidized to dihydrofolate. Dihydrofolate is rednced to tetrahydrofolate via dihydrofolate reductase so more methylene If, A °-tetrahydrofolate is made from serine in a reaction that is catalyzed by serine hydroxymethyltransferase. These three reactions, which are essential for the formation of dTMP, are shown in Fig. 14-20. 

Several processes described above use one-carbon derivatives of tetrahydrofolic acid (Fig. 14-22). E.g., the synthesis of the purine ring (Eig. 14-18) requires N °-formyl tetrahydrofolate. Thymidylate synthetase, a key enzyme in pyrimidine synthesis, uses FP,N -methylene tetrahydrofolate both as a donor of a methyl group... 

Tetrahydrofolates (THF) interconvert several one-carbon compounds or fragments. As is indicated in Fig. 18, formaldehyde released from the PLP-dependent cleavage of serine is immediately trapped by THF (Fig. 14). Nitrogen N1 adds to formaldehyde to form a carboxymethyl (—CH2—COOH) derivative which can than react reversibly with loss of water to form a cyclic adduct (Fig. 18). This compound can be oxidized to the N °-methyl form. Both of these are important intermediates in a variety of biosynthetic processes. The third one-carbon carrier is vitamin B12 which can act as an acceptor, taking the methyl group from methyl-THF to form... 

The methyl group attached to N-5 of tetrahydrofolic acid becomes transferred to S-adenosylhomocysteine and the S-adenosylmethionine thus formed is the compound that transfers methyl groups (for methyl esters and ethers, and N-methyl groups) in nature (Figure 2.16). 

Tetrahydrofolate can carry one-carbon fragments attached to N-5 (formyl, formimino, or methyl groups), N-10 (formyl group), or bridging N-5 to N-10 (methylene or methenyl groups). 5-Formyl-tetrahydrofolate is more stable than folate and is therefore used pharma-... 

Although tetrahydrofolate can carry a methyl group at N-5, the transfer potential of this methyl group is insufficient for most biosynthetic reactions. S -Adenosyl-methionine (adoMet) is the preferred cofactor for biological methyl group transfers. It is synthesized from ATP and methionine by the action of methionine... 

Phosphorylation of dCDP to dCTP (step k, Fig. 25-14) completes the biosynthesis of the first of the pyrimidine precursors of DNA. The uridine nucleotides arise in two ways. Reduction of UDP yields dUDP (step), Fig. 25-14). However, the deoxycytidine nucleotides are more often hydrolytically deaminated (reactions / and / ) 274 Methylation of dUMP to form thymidylate, dTMP (step n, Fig. 25-14), is catalyzed by thymidylate synthase. The reaction involves transfer of a 1-carbon unit from methylene tetrahydrofolic acid with subsequent reduction using THF as the electron donor. A probable mechanism is shown in Fig. 15-21. See also Box 15-E. Some bacterial transfer RNAs contain 4-thiouridine (Fig. 5-33). The sulfur atom is introduced by a sulfurtransferase (the Thil gene product in E. coli). The same protein is essential for thiamin biosynthesis (Fig. 25-21)274a... 

The methyl donor is methylene-tetrahydrofolate. The reaction involves formation of a methylene bridge between N-5 of the coenzyme and C-5 of dUMI) followed by transfer of hydrogen from the pyrazine ring of tetrahydrofolate,... 

The one-carbon group carried by tetrahydrofolate is bonded to its N-5 or N-10 nitrogen atom (denoted as TV and TV i ) or to both. This unit can exist in three oxidation states (Table 24.2). The most-reduced form carries a methyl group, whereas the intermediate form carries a methylene group. More-oxidized forms carry a formyl, formimino, or methenyl group. The fully oxidized one-carbon unit, CO2, is carried by biotin rather than by tetrahydrofolate. 

These tetrahydrofolate derivatives serve as donors of one-carbon units in a variety of biosyntheses. Methionine is regenerated from homocysteine by transfer of the methyl group ofF -methyltetrahydrofolate, as will be discussed shortly. We shall see in Chapter 25 that some of the carhon atoms of purines are acquired from derivatives of N lO-formyltetrahydrofolate. The methyl group of thymine, a pyrimidine, comes from N, N lO-methylenetetrahydrofolate. This tetrahydrofolate derivative can also donate a one-carhon unit in an alternative synthesis of glycine that starts with CO2 and NH4 +, a reaction catalyzed by glycine synthase (called the glycine cleavage enzyme when it operates in the reverse direction). 

Tetrahydrofolate can carry a methyl group on its N-5 atom, hut its transfer potential is not sufficiently high for most biosynthetic methylations. Rather, the activated methyl donor is usually S-adenosylmethionine (SAM), which is synthesized by the transfer of an adenosyl group from ATP to the sulfur atom of methionine. 

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