Redundancy in the pathway for redox regulation of mammalian methionine synthase: reductive activation by the dual flavoprotein,novel reductase 1

Methionine synthase is an essential cobalamin-dependent enzyme in mammals that catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to give tetrahydrofolate and methionine. It is oxidatively labile and requires for its sustained activity an auxiliary repair system that catalyzes a reductive methylation reaction. Genetic and biochemical studies have demonstrated that the soluble dual flavoprotein oxidoreductase, methionine synthase reductase, serves as a redox partner for methionine synthase in an NADPH-dependent reaction. However, three reports suggest the possibility of redundancy in this redox pathway. First, a hyperhomocysteinemic patient has been reported who has an isolated functional deficiency of methionine synthase but appears to be distinct from the cblE and cblG classes of patients with defects in methionine synthase reductase and methionine synthase, respectively. Second, another dual flavoprotein oxidoreductase with significant homology to methionine synthase reductase, NR1, has been described recently, but its function is unknown. Third, methionine synthase can be activated in vitro by a two-component redox system comprised of soluble cytochrome b5 and P450 reductase. In this study, we demonstrate a function for human NR1 in vitro. It is able to fully activate methionine synthase in the presence of soluble cytochrome b5 with a Vmax of 2.8 +/- 0.1 micromol min(-1) mg(-1) protein, which is comparable with that seen with methionine synthase reductase. The K(actNR1) is 1.27 +/- 0.16 microm, and a 20-fold higher stoichiometry of reductase to methionine synthase is required for NR1 versus methionine synthase reductase, suggesting that it may represent a minor pathway in the cell, assuming that the two proteins are present at similar levels.     

Interaction of flavodoxin with cobalamin-dependent methionine synthase

Cobalamin-dependent methionine synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine, forming tetrahydrofolate and methionine. The Escherichia coli enzyme, like its mammalian homologue, is occasionally inactivated by oxidation of the cofactor to cob(II)alamin. To return to the catalytic cycle, the cob(II)alamin forms of both the bacterial and mammalian enzymes must be reductively remethylated. Reduced flavodoxin donates an electron for this reaction in E. coli, and S-adenosylmethionine serves as the methyl donor. In humans, the electron is thought to be provided by methionine synthase reductase, a protein containing a domain with a significant degree of homology to flavodoxin. Because of this homology, studies of the interactions between E. coli flavodoxin and methionine synthase provide a model for the mammalian system. To characterize the binding interface between E. coli flavodoxin and methionine synthase, we have employed site-directed mutagenesis and chemical cross-linking using carbodiimide and N-hydroxysuccinimide. Glutamate 61 of flavodoxin is identified as a cross-linked residue, and lysine 959 of the C-terminal activation domain of methionine synthase is assigned as its partner. The mutation of lysine 959 to threonine results in a diminished level of cross-linking, but has only a small effect on the affinity of methionine synthase for flavodoxin. Identification of these cross-linked residues provides evidence in support of a docking model that will be useful in predicting the effects of mutations observed in mammalian homologues of E. coli flavodoxin and methionine synthase.     

Differences in the efficiency of reductive activation of methionine synthase and exogenous electron acceptors between the common polymorphic variants of human methionine synthase reductase

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.     

Electron transfer in human methionine synthase reductase studied by stopped-flow spectrophotometry

Human methionine synthase reductase (MSR) is a key enzyme in folate and methionine metabolism as it reactivates the catalytically inert cob(II)alamin form of methionine synthase (MS). Electron transfer from MSR to the cob(II)alamin cofactor coupled with methyl transfer from S-adenosyl methionine returns MS to the active methylcob(III)alamin state. MSR contains stoichiometric amounts of FAD and FMN, which shuttle NADPH-derived electrons to the MS cob(II)alamin cofactor. Herein, we have investigated the pre-steady state kinetic behavior of the reductive half-reaction of MSR by anaerobic stopped-flow absorbance and fluorescence spectroscopy. Photodiode array and single-wavelength spectroscopy performed on both full-length MSR and the isolated FAD domain enabled assignment of observed kinetic phases to mechanistic steps in reduction of the flavins. Under single turnover conditions, reduction of the isolated FAD domain by NADPH occurs in two kinetically resolved steps: a rapid (120 s(-1)) phase, characterized by the formation of a charge-transfer complex between oxidized FAD and NADPH, is followed by a slower (20 s(-1)) phase involving flavin reduction. These two kinetic phases are also observed for reduction of full-length MSR by NADPH, and are followed by two slower and additional kinetic phases (0.2 and 0.016 s(-1)) involving electron transfer between FAD and FMN (thus yielding the disemiquinoid form of MSR) and further reduction of MSR by a second molecule of NADPH. The observed rate constants associated with flavin reduction are dependent hyperbolically on NADPH and [4(R)-2H]NADPH concentration, and the observed primary kinetic isotope effect on this step is 2.2 and 1.7 for the isolated FAD domain and full-length MSR, respectively. Both full-length MSR and the separated FAD domain that have been reduced with dithionite catalyze the reduction of NADP+. The observed rate constant of reverse hydride transfer increases hyperbolically with NADP+ concentration with the FAD domain. The stopped-flow kinetic data, in conjunction with the reported redox potentials of the flavin cofactors for MSR [Wolthers, K. R., Basran, J., Munro, A. W., and Scrutton, N. S. (2003) Biochemistry, 42, 3911-3920], are used to define the mechanism of electron transfer for the reductive half-reaction of MSR. Comparisons are made with similar stopped-flow kinetic studies of the structurally related enzymes cytochrome P450 reductase and nitric oxide synthase.     

Tryptophan 697 modulates hydride and interflavin electron transfer in human methionine synthase reductase

Human methionine synthase reductase (MSR), a diflavin oxidoreductase, plays a vital role in methionine and folate metabolism by sustaining methionine synthase (MS) activity. MSR catalyzes the oxidation of NADPH and shuttles electrons via its FAD and FMN cofactors to inactive MS-cob(II)alamin. A conserved aromatic residue (Trp697) positioned next to the FAD isoalloxazine ring controls nicotinamide binding and catalysis in related flavoproteins. We created four MSR mutants (W697S, W697H, S698Δ, and S698A) and studied their associated kinetic behavior. Multiwavelength stopped-flow analysis reveals that NADPH reduction of the C-terminal Ser698 mutants occurs in three resolvable kinetic steps encompassing transfer of a hydride ion to FAD, semiquinone formation (indicating FAD to FMN electron transfer), and slow flavin reduction by a second molecule of NADPH. Corresponding experiments with the W697 mutants show a two-step flavin reduction without an observable semiquinone intermediate, indicating that W697 supports FAD to FMN electron transfer. Accelerated rates of FAD reduction, steady-state cytochrome c(3+) turnover, and uncoupled NADPH oxidation in the S698Δ and W697H mutants may be attributed to a decrease in the energy barrier for displacement of W697 by NADPH. Binding of NADP(+), but not 2',5'-ADP, is tighter for all mutants than for native MSR. The combined studies demonstrate that while W697 attenuates hydride transfer, it ensures coenzyme selectivity and accelerates FAD to FMN electron transfer. Moreover, analysis of analogous cytochrome P450 reductase (CPR) variants points to key differences in the driving force for flavin reduction and suggests that the conserved FAD stacking tryptophan residue in CPR also promotes interflavin electron transfer.     

Impeded electron transfer from a pathogenic FMN domain mutant of methionine synthase reductase and its responsiveness to flavin supplementation

Methionine synthase reductase (MSR) is a diflavin oxidoreductase that transfers electrons from NADPH to oxidized cobalamin and plays a vital role in repairing inactive cobalamin-dependent methionine synthase. MSR deficiency is a recessive genetic disorder affecting folate and methionine metabolism and is characterized by elevated levels of plasma homocysteine. In this study, we have examined the molecular basis of MSR dysfunction associated with a patient mutation, A129T, which is housed in the FMN binding domain and is adjacent to a cluster of conserved acidic residues found in diflavin oxidoreductases. We show that the substitution of alanine with threonine destabilizes FMN binding without affecting the NADPH coenzyme specificity or affinity, indicating that the mutation's effects may be confined to the FMN module. The A129T MSR mutant transfers electrons to ferricyanide as efficiently as wild type MSR but the rate of cytochrome c, 2,6-dichloroindophenol, and menadione reduction is decreased 10-15 fold. The mutant is depleted in FMN and reactivates methionine synthase with 8% of the efficiency of wild type MSR. Reconstitution of A129T MSR with FMN partially restores its ability to reduce cytochrome c and to reactivate methionine synthase. Hydrogen-deuterium exchange mass spectrometric studies localize changes in backbone amide exchange rates to peptides in the FMN-binding domain. Together, our results reveal that the primary biochemical penalty associated with the A129T MSR mutant is its lower FMN content, provide insights into the distinct roles of the FAD and FMN centers in human MSR for delivering electrons to various electron acceptors, and suggest that patients harboring the A129T mutation may be responsive to riboflavin therapy.     

Quantitation of rate enhancements attained by the binding of cobalamin to methionine synthase

Cobalamin-dependent methionine synthase (MetH) catalyzes the methylation of homocysteine using methyltetrahydrofolate as the methyl donor. The cobalamin cofactor serves as an intermediate carrier of the methyl group from methyltetrahydrofolate to homocysteine. In the two half-reactions that comprise turnover for MetH, the cobalamin is alternatively methylated by methyltetrahydrofolate and demethylated by homocysteine to form methionine. Upon binding to the protein, the usual dimethylbenzimidazole ligand is replaced by the imidazole side chain of His759 [Drennan, C. L., Huang, S., Drummond, J. T., Matthews, R. G., and Ludwig, M. L. (1994) Science 266, 1669-1674]. Despite the ligand replacement that accompanies binding of cobalamin to the holo-MetH protein, a MetH(2-649) fragment of methionine synthase that contains the regions that bind homocysteine and methyltetrahydrofolate utilizes exogenously supplied cobalamin in methyl transfer reactions akin to those of the catalytic cycle. However, the interactions of MetH(2-649) with endogenous cobalamin are first order in cobalamin, while the half-reactions catalyzed by the holoenzyme are zero order in cobalamin, so rate constants for reactions of bound and exogenous cobalamins cannot be compared. In this paper, we investigate the catalytic rate enhancements generated by binding cobalamin to MetH after dividing the protein in half and reacting MetH(2-649) with a second fragment, MetH(649-1227), that harbors the cobalamin cofactor. The second-order rate constant for demethylation of methylcobalamin by Hcy is elevated 60-fold and that for methylation of cob(I)alamin is elevated 120-fold. Thus, binding of cobalamin to MetH is essential for efficient catalysis.     

Cloning and identification of methionine synthase gene from Pichia pastoris

Methionine synthase (MS) is grouped into two classes. Class One MS (MetH) and Class Two MS (MetE) share no homology and differ in their catalytic model. Based on the conserved sequences of metE genes from different organisms, a segment of the metE gene was first cloned from Pichia pastoris genomic DNA by PCR, and its 5‘ and 3‘ regions were further cloned by 5‘- and 3‘-rapid amplification of cDNA ends (RACE), respectively. The assembled sequence reveals an open reading frame encoding a polypeptide of 768 residues, and the deduced product shares 76% identity with MetE of Saccharomyces cerevisiae. P. pastoris methionine synthase (PpMetE) consists of two domains common to MetEs. The active site is located in the C-terminal domain, in which the residues involved in the interaction of zinc with substrates are conserved. Homologous expression of PpMetE in P. pastoris was achieved, and the heterologous expression of PpMetE in the S. cerevisiae strain XJB3-1D that is MetE-defective restored the growth of the mutant on methionine-free minimal media. The gene sequence has been submitted to GenBank/EMBL/DDBJ under accession No. AY601648.     

Kinetic and thermodynamic characterization of the common polymorphic variants of human methionine synthase reductase

Human methionine synthase reductase (MSR) is a protein containing both FAD and FMN, and it reactivates methionine synthase that has lost activity due to oxidation of cob(I)alamin to cob(II)alamin. In this study, anaerobic redox titrations were employed to determine the midpoint reduction potentials for the flavin cofactors in two highly prevalent polymorphic variants of MSR, I22/L175 and M22/S175. The latter is a genetic determinant of plasma homocysteine levels and has been linked to premature coronary artery disease, Down's syndrome, and neural tube defects. The I22/L175 polymorphism has been described in a homocystinuric patient. Interestingly, this polymorphism is in the extended linker region between the two flavin domains, which may mediate or facilitate interaction with methionine synthase. In MSR I22/L175, the FMN potentials are -103 mV (oxidized/semiquinone) and -175 mV (semiquinone/hydroquinone) at pH 7.0 and 25 degrees C, and the corresponding FAD potentials are -252 and -285 mV, respectively. For the M22/S175 variants, the values of the four midpoint potentials are -114 mV (FMN oxidized/semiquinone), -212 mV (FMN semiquinone/hydroquinone), -236 mV (FAD oxidized/semiquinone), and -264 mV (FAD semiquinone/hydroquinone). The midpoint potential values in the two variants are generally comparable to those originally determined for the MSR I22/S175 variant [Wolthers, K. R. (2003) Biochemistry 42, 3911-3920], with relatively minor variations in the different redox couples. In each case, blue neutral flavin semiquinone species are stabilized on both flavins, and are characterized by a broad absorption band in the long wavelength region. In addition, stopped-flow absorption and fluorescence spectroscopy were used to study the pre-steady state reduction kinetics by NADPH of the two polymorphic variants. The reversible kinetic model proposed for wild-type MSR was validated for the I22/L175 and M22/S175 variants. Thus, the biochemical penalties associated with these polymorphisms, which result in less effective methionine synthase activation, do not appear to result from differences in their reduction kinetics. It is likely that differences in their relative affinities for the redox partner, methionine synthase, underlie the differences in the relative efficiencies of reductive activation exhibited by the variants.     

Purification and properties of chorismate synthase from Bacillus subtilis.

Chorismatic synthase was purified to apparent homogeneity from Bacillus subtilis. The enzyme required NADPH-dependent flavin reductase, Mg2+, NADPH, and flavin (FMN or FAD) for activity. The molecular weight of chorismate synthase was 24,000 as determined by sodium dedecyl sulfate (SDS)-gel electrophoresis. The enzyme was also isolated in a complex form associated with NADPH-dependent flavin reductase and another enzyme of the aromatic amino acid pathway, dehydroquinate synthase. On SDS-gel electrophoresis, this form was resolved into three bands with molecular weights of 13,000, 17,000, and 24,000. The enzyme complex was easily dissociated and the dissociation resulted in a change in the chromatographic properties of NADPH-dependent flavin reductase which was no longer retained on phosphocellulose whereas chorismate synthase was still adsorbed. Chorismate synthase activity was linear with time and protein concentration, whereas partially purified preparations showed a significant lag period before the reaction took place. Moreover, crude or partially purified enzyme preparations were completely inactivated by dilution and the activity could be recovered by addition of flavin reductase. A possible role of NADPH-dependent flavin reductase in the activation and regulation of chorismate synthase activity is discussed.     

Purification and characterization of NADPH-dependent flavin reductase. An enzyme required for the activation of chorismate synthase in Bacillus subtilis.

NADPH-dependent flavin reductase (required for the activation of chorismate synthase) was purified to homogeneity from cell-free extracts of Bacillus subtilis. The enzyme has a molecular weight of 13,000 as determined by sodium dodecyl sulfate-gel electrophoresis, is specific for NADPH, and requires a divalent metal ion and either FMN or FAD for maximal rates of NADPH oxidation. The enzyme is able to reduce 2,6-dichlorophenolindophenol (DCIP) in the presence of NADPH and a divalent metal ion. Both catalytic activities were completely inhibited by EDTA. The Km for FMN is 1.25 X 10(-5) M and for NADPH 7.8 X 10(-5) M with oxygen as the final electron acceptor, and 3.85 X 10(-4) M with DCIP as the final electron acceptor. The enzyme was also isolated in association with chorismate synthase and dehydroquinate synthase. The enzyme associated with the complex has the same catalytic properties as the dissociated enzyme except that it requires both a divalent metal ion and FMN for DCIP reduction. Maximal enzyme activity was observed when the enzyme was preincubated with FMN and the divalent metal ion. The enzyme complex is easily dissociable and the dissociation of the enzyme complex resulted in the failure of NADPH-dependent flavin reductase to adsorb to phosphocellulose.     

Targeted disruption of the methionine synthase gene in mice

Alterations in homocysteine, methionine, folate, and/or B12 homeostasis have been associated with neural tube defects, cardiovascular disease, and cancer. Methionine synthase, one of only two mammalian enzymes known to require vitamin B12 as a cofactor, lies at the intersection of these metabolic pathways. This enzyme catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine, generating tetrahydrofolate and methionine. Human patients with methionine synthase deficiency exhibit homocysteinemia, homocysteinuria, and hypomethioninemia. They suffer from megaloblastic anemia with or without some degree of neural dysfunction and mental retardation. To better study the pathophysiology of methionine synthase deficiency, we utilized gene-targeting technology to inactivate the methionine synthase gene in mice. On average, heterozygous knockout mice from an outbred background have slightly elevated plasma homocysteine and methionine compared to wild-type mice but seem to be otherwise indistinguishable. Homozygous knockout embryos survive through implantation but die soon thereafter. Nutritional supplementation during pregnancy was unable to rescue embryos that were completely deficient in methionine synthase. Whether any human patients with methionine synthase deficiency have a complete absence of enzyme activity is unclear. These results demonstrate the importance of this enzyme for early development in mice and suggest either that methionine synthase-deficient patients have residual methionine synthase activity or that humans have a compensatory mechanism that is absent in mice.     

Biochemical and structural characterization of Pseudomonas aeruginosa Bfd and FPR: ferredoxin NADP+ reductase and not ferredoxin is the redox partner of heme oxygenase under iron-starvation conditions

Among the 118 genes upregulated by Pseudomonas aeruginosa in response to iron starvation [Ochsner, U. A., Wilderman, P. J., Vasil, A. I., and Vasil, M. L. (2002) Mol. Microbiol. 45, 1277-1287], we focused on the products of the two genes encoding electron transfer proteins, as a means of identifying the redox partners of the heme oxygenase (pa-HO) expressed under low-iron stress conditions. Biochemical and spectroscopic investigations demonstrated that the bfd gene encodes a 73-amino acid protein (pa-Bfd) that incorporates a [2Fe-2S]2+/+ center, whereas the fpr gene encodes a 258-residue NADPH-dependent ferredoxin reductase (pa-FPR) that utilizes FAD as a cofactor. In vitro reconstitution of pa-HO catalytic activity with the newly characterized proteins led to the surprising observation that pa-FPR efficiently supports the catalytic cycle of pa-HO, without the need of a ferredoxin. In comparison, electron transfer from pa-Bfd to pa-HO is sluggish, which strongly argues against the possibility that the seven electrons needed by pa-HO to degrade biliverdin are transferred from NADPH to pa-HO in a ferredoxin (Bfd)-dependent manner. Given that pa-HO functions to release iron from exogenous heme acquired under iron-starvation conditions, the use of a flavoenzyme rather than an iron-sulfur center-containing protein to support heme degradation is an efficient use of resources in the cell. The crystal structure of pa-FPR (1.6 A resolution) showed that its fold is comparable that of the superfamily of ferredoxin reductases and most similar to the structure of Azotobacter vinelandii FPR and Escherichia coli flavodoxin reductase. The latter two enzymes interact with distinct redox partners, a ferredoxin and a flavodoxin, respectively. Hence, findings reported herein extend the range of redox partners recognized by the fold of pa-FPR to include a heme oxygenase (pa-HO).     

A new and suitable reconstructed system for NADPH-dependent microsomal lipid peroxidation

In order to evaluate the O-2 participation in NADPH-dependent microsomal lipid peroxidation, we used reconstructed system which contained detergent-solubilized NADPH-dependent cytochrome P-450 reductase, cytochrome P-450, phospholipid liposomes, NADPH and Fe3+-ADP. Lipid peroxidation, monitored by the formation of thiobarbituric acid-reactive substance, was increased with increasing concentration of detergent-solubilized NADPH cytochrome P-450 reductase, cytochrome P-450 or Fe3+-ADP. Cytochrome P-450-dependent lipid peroxidation was parallel to O-2 generation monitored by chemiluminescence probe with 2-methyl-6-(p-methoxyphenol)-3,7-dihydroimidazo[1,2-a]pyrazin++ +-3-one. Lipid peroxidation was significantly inhibited by superoxide dismutase, but not by catalase or sodium benzoate. The reconstructed system herein described is considered to be very close to NADPH-dependent microsomal lipid peroxidation system.     

Electron acceptor specificity of ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli

Reduced flavodoxin I (Fld1) is required in Escherichia coli for reductive radical generation in AdoMet-dependent radical enzymes and reductive activation of cobalamin-dependent methionine synthase. Ferredoxin (Fd) and flavodoxin II (Fld2) are also present, although their precise roles have not been ascertained. Ferredoxin (flavodoxin):NADP+ oxidoreductase (FNR) was discovered in E. coli as an NADPH-dependent reductant of Fld1 that facilitated generation of active methionine synthase in vitro; FNR and Fld1 will also supply electrons for the reductive cleavage of AdoMet essential for generating protein or substrate radicals in pyruvate formate-lyase, class III ribonucleotide reductase, biotin synthase, and, potentially, lipoyl synthase. As part of ongoing efforts to understand the various redox pathways that will support AdoMet-dependent radical enzymes in E. coli, we have examined the relative specificity of E. coli FNR for Fd, Fld1, and Fld2. While FNR will reduce all three proteins, Fd is the kinetically and thermodynamically preferred partner. Fd binds to FNR with high affinity (K(d)相似文献   

Activation of class III ribonucleotide reductase by flavodoxin: a protein radical-driven electron transfer to the iron-sulfur center

In its active form, Escherichia coli class III ribonucleotide reductase homodimer alpha(2) relies on a protein free radical located on the Gly(681) residue of the alpha polypeptide. The formation of the glycyl radical, namely, the activation of the enzyme, involves the concerted action of four components: S-adenosylmethionine (AdoMet), dithiothreitol (DTT), an Fe-S protein called beta or "activase", and a reducing system consisting of NADPH, NADPH:flavodoxin oxidoreductase, and flavodoxin (fldx). It has been proposed that a reductant serves to generate a reduced [4Fe-4S](+) cluster absolutely required for the reductive cleavage of AdoMet and the generation of the radical. Here, we suggest that the one-electron reduced form of flavodoxin (SQ), the only detectable product of the in vitro enzymatic reduction of flavodoxin, can support the formation of the glycyl radical. However, the redox potential of the Fe-S center of the enzyme is shown to be approximately 300 mV more negative than that of the SQ/fldx couple and not shifted to a more positive value by AdoMet binding. It is also more negative than that of the HQ/SQ couple, HQ being the fully reduced form of flavodoxin. Our interpretation is that activation of ribonucleotide reductase occurs through coupling of the reduction of the Fe-S center by flavodoxin to two thermodynamically favorable reactions, the oxidation of the cluster by AdoMet, yielding methionine and the 5'-deoxyadenosyl radical, and the oxidation of the glycine residue to the corresponding glycyl radical by the 5'-deoxyadenosyl radical. The second reaction plays the major role on the basis that a Gly-to-Ala mutation results in a greatly decreased production of methionine.     

Heterogeneity in cblG: differential retention of cobalamin on methionine synthase.

Cultured fibroblasts from patients with functional methionine synthase deficiency have been shown to belong to two complementation classes, cblE and cblG. Both are associated with decreased intracellular levels of methylcobalamin (MeCbl) and decreased incorporation of label from 5-methyltetrahydrofolate into macromolecules. Methionine synthase specific activity is normal or near normal in cell extracts from cblE patients under standard reducing conditions, whereas specific activity is low in cblG extracts. Seven of 10 cblG cell lines accumulated [57Co]CN-Cbl equivalent to control cells and showed similar proportions of label associated with the two intracellular cobalamin binders, methionine synthase and methylmalonyl-CoA mutase. The remaining three cblG lines showed reduced accumulation of labeled Cbl and virtually none associated with methionine synthase. The specific activity of methionine synthase was decreased in cell extracts from both cblG subgroups, being almost undetectable in extracts from the latter three lines. Incorporation of label from [14C]MeTHF into either macromolecules or into methionine was decreased in both cblG groups, but was paradoxically higher in the three lines with very low in vitro methionine synthase activity. These results demonstrate further heterogeneity within cblG and suggest that the defect in the three variant lines affects the ability of methionine synthase to retain Cbl.     

基于蛋氨酸代谢调控的内源性抗氧化分子机制

蛋氨酸是一种含硫必需氨基酸,在蛋白质组成和新陈代谢中都发挥着独特的作用。蛋氨酸具有内源性抗氧化作用,一方面蛋氨酸在蛋氨酸亚砜还原酶(MSR)作用下通过自身氧化还原反应来发挥内源性抗氧化作用,另一方面蛋氨酸可通过代谢途径(GSH合成、Nrf2抗氧化通路等)来增强内源性抗氧化能力。然而目前缺乏对蛋氨酸抗氧化分子机制全面深入的研究报道。因此,本文在蛋氨酸代谢的基础上,对蛋氨酸促进GSH合成、激活MSR抗氧化系统以及调控Nrf2抗氧化通路的分子机制进行综述,并对GSH合成、MSR与Nrf2抗氧化体系之间的关系进行阐述,为全面解析蛋氨酸内源性抗氧化分子机制提供理论依据。