Methylene-Tetrahydrofolate Reductase

5-formyl-tetrahydrofolate undergoes nonenzymic cyclization to methenyl-tetrahydrofolate in the acid conditions of the stomach. 

Methylene-tetrahydrofolate reductase is inhibited by S-adenosyhnethi-onine, which inhibits reduction of the flavin prosthetic group by NADPH. S-Adenosylhomocysteine overcomes this inhibition to some extent, as might be expected for an enzyme that is indirectly involved in the regulation of methionine and S-adenosylmethionine concentrations in the cell. 

Kang and coworkers (1991) reported a variant of methylene-tetrahydrofolate reductase, in which cytosine is replaced by thymidine, resulting in a change of alanine to valine, in people who were hyperhomocysteinemic (Section 10.3.4.2). The variant enzyme is thermolabile (i.e., it is unstable to heating to about 40° to 45°C), and subjects who are homozygous for the thermolabile enzyme have about 50% of normal enzyme activity in tissues. Not only is the enzyme labile on moderate heating in vitro, but it is also unstable in 

Most dietary folate is reduced and methylated to methyi-tetrahydro-folate in the intestineil mucosa (Section 10.2.1). Intestinal mucosal cells have a rapid turnover, typicedly 48 hours from proliferation in the crypt to shedding at the tip of the villus. This means that an unstable vcuiant of the enzyme, which loses acdvity over a shorter time thcui the normal enzyme, is probably irrelevant in cells that have such a rapid turnover. A high inttike of folate would therefore result in a relatively high rate of supply of methyl-teticihydrofolate to cells, arising from newly absorbed folate, so that impeured turnover of folate within cells would be less important. 

Methylene-tetrahydrofolate reductase is a flavoprotein. There is some evidence that riboflavin also stabilizes the thermolabile variant tmd that riboflavin supplements may lower plasma homocysteine in people who tue homozygous for the vtiriant enzyme (McNulty et al., 2002). 

There is a second polymorphism of methylene-tetrahydrofolate reductase in about 10% of the population, in which adenosine is replaced by cytosine. 

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Supplements of 400 Ig/d of folate begun before conception result in a significant reduction in the incidence of neural mbe defects as found in spina bifida. Elevated blood homocysteine is an associated risk factor for atherosclerosis, thrombosis, and hypertension. The condition is due to impaired abihty to form methyl-tetrahydrofolate by methylene-tetrahydrofolate reductase, causing functional folate deficiency and resulting in failure to remethylate homocysteine to methionine. People with the causative abnormal variant of methylene-tetrahydrofolate reductase do not develop hyperhomocysteinemia if they have a relatively high intake of folate, but it is not yet known whether this affects the incidence of cardiovascular disease. 

Frosst P, Blom HJ, Milos R et al. A candidate genetic risk factor for vascular disease a common mutation in methylene-tetrahydrofolate reductase. Nature Genet 1995 10 111-113. 

Mucositis (toxicity) from methotrexate (methylene tetrahydrofolate reductase TT variant)... 

MTHFR = methylene tetrahydrofolate reductase DHFR = dihydrofolate reductase SAM = S-adenosyl methionine... 

Fig. 14.1 Cellular pathway of methotrexate. ABCBl, ABCCl-4, ABC transporters ADA, adenosine deaminase ADP, adenosine diphosphate AICAR, aminoimidazole carboxamide ribonucleotide AMP, adenosine monophosphate ATIC, AICAR transformylase ATP, adenosine triphosphate SjlO-CH -THF, 5,10-methylene tetrahydrofolate 5-CHj-THF, 5-methyl tetrahydro-folate DHFR, dihydrofolate reductase dTMP, deoxythymidine monophosphate dUMP, deoxy-uridine monophosphate FAICAR, 10-formyl AICAR FH, dihydrofolate FPGS, folylpolyglutamyl synthase GGH, y-glutamyl hydrolase IMP, inosine monophosphate MTHFR, methylene tetrahydrofolate reductase MTR, methyl tetrahydrofolate reductase MTX-PG, methotrexate polyglutamate RFCl, reduced folate carrier 1 TYMS, thymidylate synthase. Italicized genes have been targets of pharmacogenetic analyses in studies published so far. (Reproduced from ref. 73 by permission of John Wiley and Sons Inc.)...

He isoleucine, Kyn kynurenine, Leu leucine, Lys lysine, Met methionine, MTHFR 5,10-methylene tetrahydrofolate reductase, Orn ornithine, p plasma, P5C pyrroline-5-carboxylic acid, PEA phosphoethanolamine, Phe phenylalanine, P-Hyl O-phosphohydroxylysine, Pip pipecolic acid, Pro proline, Sacch saccharopine, Sar sarcosine, Ser serine,... 

CFD is further associated with the following inherited metabolic disorders 5,10-methylen-tetrahydrofolate reductase (MTHFR) deficiency [7], 3-phos-phoglycerate dehydrogenase (PGDH) deficiency [8], dihydropteridine reductase (DHPR) deficiency [9], as well as with Rett syndrome [10], and Aicardi-Gou res Syndrome [11]. Furthermore, folate deficiency may be associated with congenital folate malabsorption, severe malnutrition, and formiminotransferase deficiency. 

Kaye JM, Stanton KG, McCann VJ, Vasikaran VB, Taylors RR, van Bockxmeer FM. 2002. Homocysteine, folate, methylene tetrahydrofolate reductase genotype and vascular morbidity in diabetic subjects. Clin Sci 102 631-637. 

Riboflavin (vitamin B2) Folate cycle reduction of 5,10-methyltetrahydrofolate cofactor for methylene-tetrahydrofolate reductase... 

In the folate coenzymes, the pteridine ring is fully reduced to tetrahydro-folate, although the oxidized form, dihydrofolate, is an important metabolic intermediate. In the reactions of thymidylate synthetase (Section 10.3.3) and methylene tetrahydrofolate reductase (Section 10.3.2.1), the pteridine ring has a redox role in the reaction. The folate coenzymes are conjugated with up to six additional glutamate residues, finked by y-glutamyl peptide bonds. 

Figure 10.7. Reaction of methylene-tetrahydrofolate reductase (EC 1.7.99.5). THE, tetrahydrofolate.

Methylene-Tetrahydrofolate Reductase The reduction of methylene-tetrahydrofolate to methyl-tetrahydrofolate, shown in Figure 10.7, is catalyzed hy methylene-tetrahydrofolate reductase, a flavin adenine dinucleotide-dependent enzyme during the reaction, the pteridine ring of the substrate is oxidized to dihydrofolate, then reduced to tetrahydrofolate by the flavin, which is reduced by nicotinamide adenine dinucleotide phosphate (NADPH Matthews and Daubner, 1982). The reaction is irreversible under physiological conditions, and methyl-tetrahydrofolate - which is the main form of folate taken up into tissues (Section 10.2.2) - can only be utilized after demethylation catalyzed by methionine synthetase (Section 10.3.4). 

This functional deficiency of folate is exacerbated by the associated low concentrations of methionine and S-adenosyl methioitine, although most tissues (apart from the central nervous system) also have betaine-homocysteine methyltransferase that may be adequate to maintain tissue pools of methionine. Under normal conditions S-adenosyl methioitine inhibits methylene-tetrahydrofolate reductase and prevents the formation of further methyl-tetrahydrofolate. Relief of this inhibition results in increased reduction of one-carbon substituted tetrahydrofolates to methyl-tetrahydrofolate. 

As shown in Figure 10.9, the overall reaction of methionine synthetase is the transfer of the methyl group from methyl-tetrahydrofolate to homocysteine. However, the enzyme also requires S-adenosyl methionine and a flavoprotein reducing system in addition to the cobalamin prosthetic group. A common polymorphism of methionine synthetase, in which aspartate is replaced by glycine, is associated with elevated plasma homocysteine in some cases, although it is less important than methylene-tetrahydrofolate reductase polymorphisms (Section 10.3.2.1 Harmon etal., 1999). 

Administration of diphenylhydantoin leads to decreased activity of methylene tetrahydrofolate reductase and an increased rate of oxidation of formyl tetrahydrofolate (increased oxidation of formate and histidine), with a fall in methylene- and methyl-tetrahydrofolate - the reverse of the effect of the methyl folate trap (Billings, 1984a, 1984b). 

In experimental animals and with isolated tissue preparations and organ cultures, the test can be refined by measuring the production of G02 from [ C]histidine in the presence and absence of added methionine. If the impairment of histidine metabolism is the result of primary folate deficiency, the addition of methionine wUl have no effect. By contrast, if the problem is trapping of folate as methyl-tetrahydrofolate, the addition of methionine will restore normal histidine oxidation as a result of restoring the inhibition of methylene-tetrahydrofolate reductase by S-adenosylmethionine and restoring the activity of 10-formyl-tetrahydrofolate dehydrogenase, thus permitting more normal folate metabolism (Section 10.3.4.1). 

Supplements of400 /xg per day of folic acid, begun before conception, halve the risk of neural tube defect (Section 10.9.4), and similar supplements reduce the plasma concentration of homocysteine in people homozygous for the ther-molabile variant of methylene-tetrahydrofolate reductase (Section 10.3.4.2), although it is not known whether or not this will reduce their risk of cardiovascular disease. A number of manufacturers voluntarily enrich foods with folic acid. In the United States and other countries, there is mandatory enrichment of cereal products with folic acid. 

There is some evidence that riboflavin status affects the stability of the ther-molabile vmiemt of methylene tetrahydrofolate reductase (Section 10.3.2.1), and that supplements of riboflavin may lower plasma homocysteine (Section... 

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