Insights into the reactivation of cobalamin-dependent methionine synthase.

Cobalamin-dependent methionine synthase (MetH) is a modular protein that catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to produce methionine and tetrahydrofolate. The cobalamin cofactor, which serves as both acceptor and donor of the methyl group, is oxidized once every approximately 2,000 catalytic cycles and must be reactivated by the uptake of an electron from reduced flavodoxin and a methyl group from S-adenosyl-L-methionine (AdoMet). Previous structures of a C-terminal fragment of MetH (MetH(CT)) revealed a reactivation conformation that juxtaposes the cobalamin- and AdoMet-binding domains.

Spectroscopic and computational characterization of the base-off forms of cob(II)alamin.

The one-electron-reduced form of vitamin B(12), cob(II)alamin (Co(2+)Cbl), is found in several essential human enzymes, including the cobalamin-dependent methionine synthase (MetH). In this work, experimentally validated electronic structure descriptions for two "base-off" Co(2+)Cbl species have been generated using a combined spectroscopic and computational approach, so as to obtain definitive clues as to how these and related enzymes catalyze the thermodynamically challenging reduction of Co(2+)Cbl to cob(I)alamin (Co(1+)Cbl). Specifically, electron paramagnetic resonance (EPR), electronic absorption (Abs), and magnetic circular dichroism (MCD) spectroscopic techniques have been employed as complementary tools to characterize the two distinct forms of base-off Co(2+)Cbl that can be trapped in the H759G variant of MetH, one containing a five-coordinate and the other containing a four-coordinate, square-planar Co(2+) center.

Oxidation of cysteine 645 of cobalamin-independent methionine synthase causes a methionine limitation in Escherichia coli.

Cobalamin-independent methionine synthase (MetE) catalyzes the final step in Escherichia coli methionine biosynthesis but is inactivated under oxidative conditions, triggering a methionine deficiency. This study demonstrates that the mutation of MetE cysteine 645 to alanine completely eliminates the methionine auxotrophy imposed by diamide treatment, suggesting that modulation of MetE activity via cysteine 645 oxidation has significant physiological consequences for oxidatively stressed cells.

Cobalamin- and corrinoid-dependent enzymes.

This chapter reviews the literature on cobalamin- and corrinoid-containing enzymes. These enzymes fall into two broad classes, those using methylcobalamin or related methylcorrinoids as prosthetic groups and catalyzing methyl transfer reactions, and those using adenosylcobalamin as the prosthetic group and catalyzing the generation of substrate radicals that in turn undergo rearrangements and/or eliminations.

Cobalamin-dependent and cobamide-dependent methyltransferases.

Methyltransferases that employ cobalamin cofactors, or their analogs the cobamides, as intermediates in catalysis of methyl transfer play vital roles in energy generation in anaerobic unicellular organisms. In a broader range of organisms they are involved in the conversion of homocysteine to methionine. Although the individual methyl transfer reactions catalyzed are simple S(N)2 displacements, the required change in coordination at the cobalt of the cobalamin or cobamide cofactors and the lability of the reduced Co(+1) intermediates introduces the necessity for complex conformational changes during the catalytic cycle.

Spectroscopic study of the cobalamin-dependent methionine synthase in the activation conformation: effects of the Y1139 residue and S-adenosylmethionine on the B12 cofactor.

The cobalamin-dependent methionine synthase (MetH) from Escherichia coli is a modular enzyme that catalyzes a methyl group transfer from methyltetrahydrofolate to homocysteine via a methylcob(III)alamin (MeCbl) intermediate, generating tetrahydrofolate and methionine (Met). Once every approximately 2000 turnovers, the cobalamin cofactor is converted to the inactive cob(II)alamin (Co(2+)Cbl) form, from which MeCbl has to be recovered for MetH to re-enter the catalytic cycle. A particularly puzzling aspect of this reactivation process is that it requires the reduction of the Co(2+)Cbl species to cob(I)alamin (Co(1+)Cbl) by flavodoxin, a reaction that would appear to be endergonic on the basis of the corresponding reduction potentials.

Methionine synthase reductase deficiency results in adverse reproductive outcomes and congenital heart defects in mice.

Low dietary folate and polymorphisms in genes of folate metabolism can influence risk for pregnancy complications and birth defects. Methionine synthase reductase (MTRR) is required for activation of methionine synthase, a folate- and vitamin B(12)-dependent enzyme. A polymorphism in MTRR (p.

A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor.

B(12)-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that is alternately methylated by methyltetrahydrofolate to form methylcobalamin and demethylated by homocysteine to form cob(I)alamin. Major domain rearrangements are required to allow cobalamin to react with three different substrates: homocysteine, methyltetrahydrofolate, and S-adenosyl-l-methionine (AdoMet). These same rearrangements appear to preclude crystallization of the wild-type enzyme.

Metal active site elasticity linked to activation of homocysteine in methionine synthases.

Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers.

Defects in homocysteine metabolism: diversity among hyperhomocyst(e)inemias.

There are now four genetic mouse models that induce hyperhomocyst(e)inemia by decreasing the activity of an enzyme involved in homocysteine metabolism: cystathionine beta-synthase, methylenetetrahydrofolate reductase, methionine synthase and methionine synthase reductase. While each enzyme deficiency leads to murine hyperhomocyst(e)inemia, the accompanying metabolic profiles are significantly and often unexpectedly, different. Deficiencies in cystathionine beta-synthase lead to elevated plasma methionine, while deficiencies of the remaining three enzymes lead to hypomethioninemia.

Ligand trans influence governs conformation in cobalamin-dependent methionine synthase.

Cobalamin-dependent methionine synthase (MetH) of Escherichia coli is a large, modular enzyme that uses a cobalamin prosthetic group as a donor or acceptor in three separate methyl transfer reactions. The prosthetic group alternates between methylcobalamin and cob(I)alamin during catalysis as homocysteine is converted to methionine using a methyl group derived from methyltetrahydrofolate. Occasional oxidation of cob(I)alamin to cob(II)alamin inactivates the enzyme.

The many flavors of hyperhomocyst(e)inemia: insights from transgenic and inhibitor-based mouse models of disrupted one-carbon metabolism.

Mouse models that perturb homocysteine metabolism, including genetic mouse models that result in deficiencies of methylenetetrahydrofolate reductase, methionine synthase, methionine synthase reductase, and cystathionine beta-synthase, and a pharmaceutically induced mouse model with a transient deficiency in betainehomocysteine methyl transferase, have now been characterized and can be compared. Although each of these enzyme deficiencies is associated with moderate to severe hyperhomocyst(e)inemia, the broader metabolic profiles are profoundly different. In particular, the various models differ in the degree to which tissue ratios of S-adenosylmethionine to S-adenosylhomocysteine are reduced in the face of elevated plasma homocyst(e)ine, and in the distribution of the tissue folate pools.

Reactivation of methionine synthase from Thermotoga maritima (TM0268) requires the downstream gene product TM0269.

The crystal structure of the Thermotoga maritima gene product TM0269, determined as part of genome-wide structural coverage of T. maritima by the Joint Center for Structural Genomics, revealed structural homology with the fourth module of the cobalamin-dependent methionine synthase (MetH) from Escherichia coli, despite the lack of significant sequence homology. The gene specifying TM0269 lies in close proximity to another gene, TM0268, which shows sequence homology with the first three modules of E.

Probing the role of the histidine 759 ligand in cobalamin-dependent methionine synthase.

Cobalamin-dependent methionine synthase (MetH) of Escherichia coli is a 136 kDa, modular enzyme that undergoes large conformational changes as it uses a cobalamin cofactor as a donor or acceptor in three separate methyl transfer reactions. At different points during the reaction cycle, the coordination to the cobalt of the cobalamin changes; most notably, the imidazole side chain of His759 that coordinates to the cobalamin in the "His-on" state can dissociate to produce a "His-off" state. Here, two distinct species of the cob(II)alamin-bound His759Gly variant have been identified and separated.

Folate Biosynthesis, Reduction, and Polyglutamylation and the Interconversion of Folate Derivatives.

Many microorganisms and plants possess the ability to synthesize folic acid derivatives de novo, initially forming dihydrofolate. All the folic acid derivatives that serve as recipients and donors of one-carbon units are derivatives of tetrahydrofolate, which is formed from dihydrofolate by an NADPH-dependent reduction catalyzed by dihydrofolate reductase (FolA). This review discusses the biosynthesis of dihydrofolate monoglutamate, its reduction to tetrahydrofolate monoglutamate, and the addition of glutamyl residues to form folylpolyglutamates.

Metabolic derangement of methionine and folate metabolism in mice deficient in methionine synthase reductase.

Hyperhomocyst(e)inemia is a metabolic derangement that is linked to the distribution of folate pools, which provide one-carbon units for biosynthesis of purines and thymidylate and for remethylation of homocysteine to form methionine. In humans, methionine synthase deficiency results in the accumulation of methyltetrahydrofolate at the expense of folate derivatives required for purine and thymidylate biosynthesis. Complete ablation of methionine synthase activity in mice results in embryonic lethality.

Integration of regulatory signals through involvement of multiple global regulators: control of the Escherichia coli gltBDF operon by Lrp, IHF, Crp, and ArgR.

Background: The glutamate synthase operon (gltBDF) contributes to one of the two main pathways of ammonia assimilation in Escherichia coli. Of the seven most-global regulators, together affecting expression of about half of all E. coli genes, two were previously shown to exert direct, positive control on gltBDF transcription: Lrp and IHF.

Global aggregation of newly translated proteins in an Escherichia coli strain deficient of the chaperonin GroEL.

In a newly isolated temperature-sensitive lethal Escherichia coli mutant affecting the chaperonin GroEL, we observed wholesale aggregation of newly translated proteins. After temperature shift, transcription, translation, and growth slowed over two to three generations, accompanied by filamentation and accretion (in approximately 2% of cells) of paracrystalline arrays containing mutant chaperonin complex. A biochemically isolated inclusion body fraction contained the collective of abundant proteins of the bacterial cytoplasm as determined by SDS/PAGE and proteolysis/MS analyses.

Human methionine synthase reductase is a molecular chaperone for human methionine synthase.

Sustained activity of mammalian methionine synthase (MS) requires MS reductase (MSR), but there have been few studies of the interactions between these two proteins. In this study, recombinant human MS (hMS) and MSR (hMSR) were expressed in baculovirus-infected insect cells and purified to homogeneity. hMSR maintained hMS activity at a 1:1 stoichiometric ratio with a K(act) value of 71 nM.

Activation of methyltetrahydrofolate by cobalamin-independent methionine synthase.

Cobalamin-independent methionine synthase (MetE) catalyzes the final step of de novo methionine synthesis using the triglutamate derivative of methyltetrahydrofolate (CH(3)-H(4)PteGlu(3)) as methyl donor and homocysteine (Hcy) as methyl acceptor. This reaction is challenging because at physiological pH the Hcy thiol is not a strong nucleophile and CH(3)-H(4)PteGlu(3) provides a very poor leaving group. Our laboratory has previously established that Hcy is ligated to a tightly bound zinc ion in the MetE active site.

Synergistic, random sequential binding of substrates in cobalamin-independent methionine synthase.

Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of the N5-methyl group of methyltetrahydrofolate (CH(3)-H(4)folate) to the sulfur of homocysteine (Hcy) to form methionine and tetrahydrofolate (H(4)folate) as products. This reaction is thought to involve a direct methyl transfer from one substrate to the other, requiring the two substrates to interact in a ternary complex. The crystal structure of a MetE.

Structural perturbations in the Ala --> Val polymorphism of methylenetetrahydrofolate reductase: how binding of folates may protect against inactivation.

In human methylenetetrahydrofolate reductase (MTHFR) the Ala222Val (677C-->T) polymorphism encodes a heat-labile gene product that is associated with elevated levels of homocysteine and possibly with risk for cardiovascular disease. Generation of the equivalent Ala to Val mutation in Escherichia coli MTHFR, which is 30% identical to the catalytic domain of the human enzyme, creates a protein with enhanced thermolability. In both human and E.

Methionine.

This review focuses on the steps unique to methionine biosynthesis, namely the conversion of homoserine to methionine. The past decade has provided a wealth of information concerning the details of methionine metabolism and the review focuses on providing a comprehensive overview of the field, emphasizing more recent findings. Details of methionine biosynthesis are addressed along with key cellular aspects, including regulation, uptake, utilization, AdoMet, the methyl cycle, and growing evidence that inhibition of methionine biosynthesis occurs under stressful cellular conditions.

Regulation of human methylenetetrahydrofolate reductase by phosphorylation.

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, the methyl donor for the conversion of homocysteine to methionine. Regulation of MTHFR activity is crucial for maintaining cellular concentrations of methionine and S-adenosylmethionine (AdoMet). Purified recombinant human MTHFR expressed in insect cells is multiply phosphorylated on an N-terminal extension of the protein that contains a highly conserved serine-rich region.