Defects in Auxiliary Redox Proteins Lead to Functional Methionine Synthase Deficiency (original) (raw)
Related papers
Biochemistry, 1996
Vitamin B 12 -dependent methionine synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine via the enzyme-bound cofactor methylcobalamin. To carry out this reaction, the enzyme must alternately stabilize six-coordinate methylcobalamin and four-coordinate cob(I)alamin oxidation states. The lower axial ligand to the cobalt in free methylcobalamin is the dimethylbenzimidazole nucleotide substituent of the corrin ring; when methylcobalamin binds to methionine synthase, this ligand is replaced by histidine 759, which in turn is linked by hydrogen bonds to aspartate 757 and thence to serine 810. We have proposed that these residues control the reactivity of the enzymebound cofactor both by increasing the coordination strength of the imidazole ligand and by allowing stabilization of cob(I)alamin via protonation of the His-Asp-Ser triad. In this paper we report results of mutation studies focusing on these catalytic residues. We have used visible absorbance spectroscopy and electron paramagnetic resonance spectroscopy to probe the coordination state of the cofactor and have used stopped-flow kinetic measurements to explore the reactivity of each mutant. We show that mutation of histidine 759 blocks turnover, while mutations of aspartate 757 or serine 810 decrease the reactivity of the methylcobalamin cofactor. In contrast, we show that mutations of these same residues increase the rate of AdoMet-dependent reactivation of cob(II)alamin enzyme. We propose that the reaction with AdoMet proceeds via a different transition state than the reactions with homocysteine and methyltetrahydrofolate. These results provide a glimpse at how a protein can control the reactivity of methylcobalamin.
Biochemistry, 1997
Methionine synthase (MetH) catalyzes the transfer of a methyl group from bound methylcobalamin to homocysteine, yielding enzyme-bound cob(I)alamin and methionine. The cofactor is then remethylated by methyltetrahydrofolate. We now demonstrate that MetH is able to catalyze methylation of free cob(I)alamin with methyltetrahydrofolate. MetH had previously been shown to catalyze methylation of homocysteine with free methylcobalamin as the methyl donor, in a reaction that is first-order in added methylcobalamin, and we have confirmed this observation using homogenous enzyme. A truncated polypeptide lacking the cobalamin-binding region of the holoenzyme, MetH(2-649), was overexpressed and purified to homogeneity. MetH(2-649) catalyzes the methylation of free cob(I)alamin by methyltetrahydrofolate and the methylation of homocysteine by free methylcobalamin. Furthermore, a protein comprising residues 2-353 of the holoenzyme has now been overexpressed and purified to homogeneity, and this protein catalyzes methyl transfer from free methylcobalamin to homocysteine but not from methyltetrahydrofolate to free cob(I)alamin. The mutations Cys310Ala and Cys311Ala in MetH(2-649) completely abolish methyl transfer from exogenous methylcobalamin to homocysteine but do not affect methyl transfer from methyltetrahydrofolate to exogenous cob(I)alamin, consistent with a modular construction for MetH. We infer that MetH is a modular protein comprising four separate regions: a homocysteine binding region (residues 2-353), a methyltetrahydrofolate binding region (residues 354-649), a region responsible for binding the cobalamin prosthetic group (residues 650-896), and an AdoMet-binding domain (residues 897-1227).
Current Opinion in Structural Biology, 1994
Cobalamin-dependent methionine synthase is a large enzyme composed of structurally and functionally distinct regions. Recent studies have begun to define the roles of several regions of the protein. In particular, the structure of a 27kDa cobalamin-binding fragment of the enzyme from Escherichia coil has been determined by X-ray crystallography, and has revealed the motifs and interactions responsible for recognition of the cofactor. The amino acid sequences of several adenosylcobalamin-dependent enzymes, the methylmalonyl coenzyme A mutases and glutamate mutases, show homology with the cobalamin-binding region of methionine synthase and retain conserved residues that are determinants for the binding of the prosthetic group, suggesting that these mutases and methionine synthase share common three-dimensional structures.
Insights into the reactivation of cobalamin-dependent methionine synthase
Proceedings of the National Academy of Sciences, 2009
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 Ϸ2,000 catalytic cycles and must be reactivated by the uptake of an electron from reduced flavodoxin and a methyl group from Sadenosyl-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. Here we describe 2 structures of a disulfide stabilized MetH CT (S-SMetH CT ) that offer further insight into the reactivation of MetH. The structure of S-SMetH CT with cob(II)alamin and Sadenosyl-L-homocysteine represents the enzyme in the reactivation step preceding electron transfer from flavodoxin. The structure supports earlier suggestions that the enzyme acts to lower the reduction potential of the Co(II)/Co(I) couple by elongating the bond between the cobalt and its upper axial water ligand, effectively making the cobalt 4-coordinate, and illuminates the role of Tyr-1139 in the stabilization of this 4-coordinate state. The structure of S-SMetH CT with aquocobalamin may represent a transient state at the end of reactivation as the newly remethylated 5-coordinate methylcobalamin returns to the 6-coordinate state, triggering the rearrangement to a catalytic conformation.
Biochemistry, 1990
The mechanism of reductive methylation of cobalamin-dependent methionine synthase (5-methyltetrahydrofo1ate:homocysteine methyltransferase, EC 2.1.1.13) has been investigated by electron paramagnetic resonance (EPR) spectroelectrochemistry. The enzyme as isolated is inactive, and its UV/visible absorbance and EPR spectra are characteristic of cob(I1)alamin. There is an absolute requirement for catalytic amounts of AdoMet and a reducing system for the formation and maintenance of active enzyme during in vitro turnover. The midpoint potentials of the enzyme-bound cob(II)alamin/cob(I)alamin and cob(III)alamin/cob(II)alamin couples have been determined to be -526 f 5 and +273 f 4 mV (versus the standard hydrogen electrode), respectively. The presence of either CH3-H,folate or AdoMet shifts the equilibrium distribution of cobalamin species observed during reduction by converting cob(1)alamin to methylcobalamin. The magnitude of these shifts is however vastly different, with AdoMet lowering the concentration of cob(1I)alamin at equilibrium by a factor of at least 3 X lo', while CH,-H,folate lowers it by a factor of 19. These studies of coupled reduction/methylation reactions elucidate the absolute requirement for AdoMet in the in vitro assay system, in which the ambient potential is approximately -350 mV versus the standard hydrogen electrode. At this potential, the equilibrium distribution of cobalamin in the presence of CH3-H4folate would be greatly in favor of the cob(1I)alamin species, whereas in the presence of AdoMet the equilibrium favors methylated enzyme. In these studies, a base-on form of cob(I1)alamin in which the dimethylbenzimidazole substituent of the corrin ring is the lower axial ligand for the cobalt has been observed for the first time on methionine synthase. Glycerol was found to influence the baseon/base-off equilibrium on the enzyme. The midpoint potential of the base-off cob(II)alamin/cob(I)alamin couple was determined to be -572 f 24 mV.
Synergistic, Random Sequential Binding of Substrates in Cobalamin-Independent Methionine Synthase †
Biochemistry, 2006
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‚CH 3 -H 4 folate binary complex shows that the methyl group is pointing away from the Hcy binding site and is quite distant from the position where the sulfur of Hcy would be, raising the possibility that this binary complex is nonproductive. The CH 3 -H 4 folate must either rearrange or dissociate before methyl transfer can occur. Therefore, determining the order of substrate binding is of interest. We have used kinetic and equilibrium measurements in addition to isotope trapping experiments to elucidate the kinetic pathway of substrate binding in MetE. These studies demonstrate that both substrate binary complexes are chemically and kinetically competent for methyl transfer and suggest that the conformation observed in the crystal structure is indeed on-pathway. Additionally, the substrates are shown to bind synergistically, with each substrate binding 30-fold more tightly in the presence of the other. Methyl transfer has been determined to be slow compared to ternary complex formation and dissociation. Simulations indicate that nearly all of the enzyme is present as the ternary complex under physiological conditions. Methionine synthases catalyze the methylation of homocysteine (Hcy) 1 by methyltetrahydrofolate (CH 3 -H 4 folate), yielding methionine and tetrahydrofolate (H 4 folate). Escherichia coli contains two genes, metH and metE, specifying two different methionine synthase enzymes that are differentially expressed (1). The MetH protein is cobalamin-dependent methionine synthase, in which the cobalamin (B 12 ) cofactor serves as an intermediary in methyl transfer, accepting a
Biochemistry, 1988
Cobalamin-dependent methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) has been isolated from Escherichia coli B in homogeneous form. The enzyme is isolated in an inactive form with the visible absorbance properties of cob(I1)alamin. The inactive enzyme exhibits an electron paramagnetic resonance (EPR) spectrum at 38 K that is characteristic of cob(1I)alamin at acid pH, where the protonated dimethylbenzimidazole substituent is not coordinated with the cobalt nucleus (base-off cobalamin). An additional, variable component of the EPR spectrum of the inactive enzyme has the characteristics of a cob(II1)alamin-superoxide complex. Scientific Societies, Tokyo] has demonstrated that the enzyme can be activated by reductive methylation using adenosylmethionine as the methyl donor. We present data indicating that the conversion of inactive to methylated enzyme is correlated with the disappearance of the EPR spectrum as expected for the conversion of paramagnetic cob(1I)alamin to diamagnetic methylcobalamin. When the methyl group is transferred from the methylated enzyme to homocysteine under aerobic conditions, cob( II)alamin/cob( 1II)alamin-superoxide enzyme is regenerated as indicated by the return of the visible absorbance properties of the initially isolated enzyme and partial return of the EPR spectrum. Our enzyme preparations contain copper in -1:l stoichiometry with cobalt as determined by atomic absorption spectroscopy. This copper is EPR silent in the enzyme as isolated and in methylated enzyme, but it can be detected as a cupric-EDTA complex following aerobic denaturation of the enzyme in the presence of mersalyl, 6 M urea, and EDTA.