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Crystal structures of cobalamin-independent methionine synthases 1. Plant enzyme -
Ferrer, J.-L., Ravanel, S., Robert, M., and Dumas, R.: Crystal structures of cobalamin-independent methionine synthase complexed with zinc, homocysteine, and methyltetrahydrofolate. Journal of Biological Chemistry 279, 44235-44238 (2004)
Cobalamin-independent methionine synthase (MetE) catalyzes the synthesis of methionine by a direct transfer of the methyl group of N5-methyltetrahydrofolate (CH3-H2PteGlun) to the sulfur atom of homocysteine (Hcy). We report here the first crystal structure of this metalloenzyme under different forms, free or complexed with the Hcy and folate substrates. The Arabidopsis thaliana MetE (AtMetE) crystals reveal a monomeric structure built by two (betaalpha)8 barrels making a deep groove at their interface. The active site is located at the surface of the C-terminal domain, facing the large interdomain cleft. Inside the active site, His647, Cys649, and Cys733 are involved in zinc coordination, whereas Asp605, Ile437, and Ser439 interact with Hcy. Opposite the zinc/Hcy binding site, a cationic loop (residues 507-529) belonging to the C-terminal domain anchors the first glutamyl residue of CH3-H4PteGlu5. The pterin moiety of CH3-H4PteGlu5 is stacked with Trp567, enabling the N5-methyl group to protrude in the direction of the zinc atom. These data suggest a structural role of the N-terminal domain of AtMetE in the stabilization of loop 507-529 and in the interaction with the poly-glutamate chain of CH3-H4PteGlun. Comparison of AtMetE structures reveals that the addition of Hcy does not lead to a direct coordination of the sulfur atom with zinc but to a reorganization of the zinc binding site with a stronger coordination to Cys649, Cys733, and a water molecule.
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2. Bacterial enzyme (Thermotoga maritima, a thermophilic bacterium)
Pejchal, R. and Ludwig, M.L.: Cobalamin-independent methionine synthase (MetE): a face-to-face double barrel that evolved by gene duplication. Public Library of Science - Biology 3, e31 (2005)
Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of a methyl group from methyltetrahydrofolate to L-homocysteine (Hcy) without using an intermediate methyl carrier. Although MetE displays no detectable sequence homology with cobalamin-dependent methionine synthase (MetH), both enzymes require zinc for activation and binding of Hcy. Crystallographic analyses of MetE from T. maritima reveal an unusual dual-barrel structure in which the active site lies between the tops of the two (betaalpha)(8) barrels. The fold of the N-terminal barrel confirms that it has evolved from the C-terminal polypeptide by gene duplication; comparisons of the barrels provide an intriguing example of homologous domain evolution in which binding sites are obliterated. The C-terminal barrel incorporates the zinc ion that binds and activates Hcy. The zinc-binding site in MetE is distinguished from the (Cys)(3)Zn site in the related enzymes, MetH and betaine-homocysteine methyltransferase, by its position in the barrel and by the metal ligands, which are histidine, cysteine, glutamate, and cysteine in the resting form of MetE. Hcy associates at the face of the metal opposite glutamate, which moves away from the zinc in the binary E.Hcy complex. The folate substrate is not intimately associated with the N-terminal barrel; instead, elements from both barrels contribute binding determinants in a binary complex in which the folate substrate is incorrectly oriented for methyl transfer. Atypical locations of the Hcy and folate sites in the C-terminal barrel presumably permit direct interaction of the substrates in a ternary complex. Structures of the binary substrate complexes imply that rearrangement of folate, perhaps accompanied by domain rearrangement, must occur before formation of a ternary complex that is competent for methyl transfer.
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Both are excellent papers, but some points remain to be explained:
Why is the reaction dependent on inorganic phosphate, and what is the mechanism?
See
Eckermann et al. (2000) for the details
 Phosphate dependence of methionine synthase activity.
The incubations were performed under standard assay conditions. Conversion rate indicates the % of the substrate converted into product. This description for the enzyme activity eliminated the problem of presenting the 90-fold difference in enzyme activity in one figure.
Eckermann, C., Eichel, J. and Schröder, J.: Plant methionine synthase: new insights into properties and expression. Biological Chemistry 381, 695-703 (2000).
We investigated methionine synthase (MSY) in Catharanthus roseus. The properties were characterized with purified protein isolated either from plant cell cultures or after heterologous expression in Escherichia coli. The protein was a monomer and accepted both the triglutamate (CH3-H4PteGlu3, apparent Km 80 µM) and the monoglutamate (CH3-H4PteGlu1, apparent Km 350 µM) of methyl-5,6,7,8-tetrahydropteroate as methyl donor, with a ratio of approximately 90 : 1 in favor of the triglutamate. Both activities required inorganic phosphate, but with different kinetics, and both were dependent on reducing agents. The activity required zinc, as shown by depletion and reconstitution experiments. Mg2+ had no effect on the activity. Two MSY isoforms purified from parsley cell cultures revealed the same properties as the C. roseus enzyme, but the parsley proteins had no detectable activity with the monoglutamate substrate. The second part of the work compared the expression of the three enzymes of the methyl cycle (MSY, S-adenosyl-L-methionine synthetase, S-adenosyl-L-homocysteine hydrolase). In cell cultures, all three enzymes were present under all conditions investigated, with small changes at the protein level and more pronounced changes at the RNA level. Studies with seedlings revealed a low expression of all three enzymes in cotyledons, when compared to hypocotyls and radiculas. Immunohistochemical experiments indicated that MSY expression in cotyledons is cell-type specific, with the strongest signals detected in the upper epidermis.
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Interesting: Location of a MetSyn in the chloroplasts -
Ravanel, S., Block, M.A., Rippert, P., Jabrin, S., Curien, G., Rebeille, F., Douce, R.: Methionine metabolism in plants: chloroplasts are autonomous for de novo methionine synthesis and can import S-adenosylmethionine from the cytosol. Journal of Biological Chemistry 279, 22548-22557 (2004).
The subcellular distribution of Met and S-adenosylmethionine (AdoMet) metabolism in plant cells discloses a complex partition between the cytosol and the organelles. In the present work we show that Arabidopsis contains three functional isoforms of vitamin B12-independent methionine synthase (MS), the enzyme that catalyzes the methylation of homocysteine to Met with 5-methyltetrahydrofolate as methyl group donor. One MS isoform is present in chloroplasts and is most likely required to methylate homocysteine that is synthesized de novo in this compartment. Thus, chloroplasts are autonomous and are the unique site for de novo Met synthesis in plant cells. The additional MS isoforms are present in the cytosol and are most probably involved in the regeneration of Met from homocysteine produced in the course of the activated methyl cycle. Although Met synthesis can occur in chloroplasts, there is no evidence that AdoMet is synthesized anywhere but the cytosol. In accordance with this proposal, we show that AdoMet is transported into chloroplasts by a carrier-mediated facilitated diffusion process. This carrier is able to catalyze the uniport uptake of AdoMet into chloroplasts as well as the exchange between cytosolic AdoMet and chloroplastic AdoMet or S-adenosylhomocysteine. The obvious function for the carrier is to sustain methylation reactions and other AdoMet-dependent functions in chloroplasts and probably to remove S-adenosylhomocysteine generated in the stroma by methyltransferase activities. Therefore, the chloroplastic AdoMet carrier serves as a link between cytosolic and chloroplastic one-carbon metabolism.
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