Methionine synthase is a key enzyme in the methionine cycle that catalyzes the transmethylation of homocysteine to methionine in a cobalamin-dependent reaction that utilizes methyltetrahydrofolate as a methyl group donor. Cob(I)alamin, a supernucleophilic form of the cofactor, is an intermediate in this reaction, and its reactivity renders the enzyme susceptible to oxidative inactivation. In bacteria, an NADPH-dependent twoprotein system comprising flavodoxin reductase and flavodoxin, transfers electrons during reactivation of methionine synthase. Until recently, the physiological reducing system in mammals was unknown. Identification of mutations in the gene encoding a putative methionine synthase reductase in the cblE class of patients with an isolated functional deficiency of methionine synthase suggested a role for this protein in activation In this study, we have cloned and expressed the cDNA encoding human methionine synthase reductase and demonstrate that it is sufficient for supporting NADPH-dependent activity of methionine synthase at a level that is comparable with that seen in the in vitro assay that utilizes artificial reductants. Methionine synthase reductase is a soluble, monomeric protein with a molecular mass of 78 kDa. It is a member of the family of dual flavoproteins and is isolated with an equimolar concentration of FAD and FMN. Reduction by NADPH results in the formation of an air stable semiquinone similar to that observed with cytochrome P-450 reductase. Methionine synthase reductase reduces cytochrome c in an NADPH-dependent reaction at a rate (0.44 mol min ؊1 mg ؊1 at 25°C) that is comparable with that reported for NR1, a soluble dual flavoprotein of unknown function, but is ϳ100-fold slower than that of P-450 reductase. The K m for NADPH is 2.6 ؎ 0.5 M, and the K act for methionine synthase reductase is 80.7 ؎ 13.7 nM for NADPH-dependent activity of methionine synthase.Homocysteine is a key junction metabolite in the methionine cycle that is formed by the hydrolysis of S-adenosylhomocysteine, the product of S-adenosylmethionine-dependent methylation reactions. Elevated levels of homocysteine are correlated with cardiovascular diseases, neural tube defects, and Alzheimer's disease (1-4). Intracellular levels of homocysteine are kept low by the action of two classes of reactions. The transmethylation reactions catalyzed by methionine synthase or betaine homocysteine methyltransferase salvage homocysteine back to the methionine cycle. Alternatively, the ␤-replacement reaction catalyzed by cystathionine ␤-synthase commits homocysteine to the trans sulfuration pathway and provides a mechanism for generating cysteine from the essential amino acid, methionine. Of the two transmethylases, betaine homocysteine methyltransferase has a relatively restricted tissue distribution, whereas methionine synthase is believed to be ubiquitous (5).The mammalian methionine synthase is a methylcobalamindependent enzyme that catalyzes the successive transfer of a methyl group from CH 3 -H 4 folate 1 to the cob...

Methionine synthase reductase (MSR) catalyzes the conversion of the inactive form of human methionine synthase to the active state of the enzyme. This reaction is of paramount physiological importance since methionine synthase is an essential enzyme that plays a key role in the methionine and folate cycles. A common polymorphism in human MSR has been identified (66A --> G) that leads to replacement of isoleucine with methionine at residue 22 and has an allele frequency of 0.5. Another polymorphism is 524C --> T, which leads to the substitution of serine 175 with leucine, but its allele frequency is not known. The I22M polymorphism is a genetic determinant for mild hyperhomocysteinemia, a risk factor for cardiovascular disease. In this study, we have examined the kinetic properties of the M22/S175 and I22/S175 and the I22/L175 and I22/S175 pairs of variants. EPR spectra of the semiquinone forms of variants I22/S175 and M22/S175 are indistinguishable and exhibit an isotropic signal at g = 2.00. In addition, the electronic absorption and reduction stoichiometries with NADPH are identical in these variants. Significantly, the variants activate methionine synthase with the same V(max); however, a 3-4-fold higher ratio of MSR to methionine synthase is required to elicit maximal activity with the M22/S175 and I22/L175 variant versus the I22/S175 enzyme. Differences are also observed between the variants in the efficacies of reduction of the artificial electron acceptors: ferricyanide, 2,6-dichloroindophenol, 3-acetylpyridine adenine dinucleotide phosphate, menadione, and the anticancer drug doxorubicin. These results reveal differences in the interactions between the natural and artificial electron acceptors and MSR variants in vitro, which are predicted to result in less efficient reductive repair of methionine synthase in vivo.

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Methylation events play a critical role in the ability of growth factors to promote normal development. Neurodevelopmental toxins, such as ethanol and heavy metals, interrupt growth factor signaling, raising the possibility that they might exert adverse effects on methylation. We found that insulin-like growth factor-1 (IGF-1)-and dopamine-stimulated methionine synthase (MS) activity and folate-dependent methylation of phospholipids in SH-SY5Y human neuroblastoma cells, via a PI3-kinase-and MAP-kinase-dependent mechanism. The stimulation of this pathway increased DNA methylation, while its inhibition increased methylationsensitive gene expression. Ethanol potently interfered with IGF-1 activation of MS and blocked its effect on DNA methylation, whereas it did not inhibit the effects of dopamine. Metal ions potently affected IGF-1 and dopamine-stimulated MS activity, as well as folate-dependent phospholipid methylation: Cu 2 þ promoted enzyme activity and methylation, while Cu þ , Pb 2 þ , Hg 2 þ and Al 3 þ were inhibitory. The ethylmercury-containing preservative thimerosal inhibited both IGF-1-and dopamine-stimulated methylation with an IC 50 of 1 nM and eliminated MS activity. Our findings outline a novel growth factor signaling pathway that regulates MS activity and thereby modulates methylation reactions, including DNA methylation. The potent inhibition of this pathway by ethanol, lead, mercury, aluminum and thimerosal suggests that it may be an important target of neurodevelopmental toxins.

Background: The diagnosis of T-cell neoplasms is often challenging, due to overlapping features with reactive T-cells and limitations of currently available T-cell clonality assays. The description of an antibody specific for one of two mutually exclusive T-cell receptor (TCR) β-chain constant regions (TRBC1) provide an opportunity to facilitate the detection of clonal TCRαβ T-cells based on TRBC-restriction. Methods: Twenty patients with mature T-cell neoplasms and 44 patients without evidence of T-cell neoplasia were studied. Peripheral blood (51), bone marrow (10), and lymph node (3) specimens were evaluated by 9-color flow cytometry including TRBC1 (