Methionine synthases catalyze the transfer of a methyl group from N5-methyl-5,6,7,8-tetrahydrofolate (CH3-H4folate) to L-homocysteine (Hcy), the terminal step in the biosynthesis of methionine. Two apparently unrelated families of proteins catalyze this reaction: cobalamin-dependent methionine synthase (MetH; EC 22.214.171.124) and cobalamin-independent methionine synthase (MetE; 5-methyltetrahydropteroyltriglutamate–homocysteine methyltransferase; EC 126.96.36.199) Organisms that synthesize or transport B12 encode the cobalamin-dependent enzyme whereas organisms that cannot obtain B12 encode only the cobalamin-independent enzyme. Escherichia coli and many other species of bacteria express both enzymes, but mammals utilize only cobalamin-dependent methionine synthase while plants and yeasts utilize only the cobalamin-independent enzyme.
MetH and MetE both face the same mechanistic challenge. They must catalyze the transfer of a very poor leaving group from the tertiary amine, CH3
folate, to a relatively poor nucleophile, the sulfur of Hcy. MetH facilitates this transfer by using cobalamin as an intermediate methyl carrier [1
]. Cobalamin accepts a methyl group from CH3
folate at one active site and donates it to Hcy at a second site [2
]. In contrast, MetE appears to catalyze the direct transfer of the methyl group from CH3
folate to Hcy [3
]. This latter strategy seems to offer a less satisfactory answer to the mechanistic problems: measured kcat
values for MetE are smaller than those for MetH by a factor of approximately 50–100.
MetE and MetH both activate Hcy by binding the thiolate form of the substrate to Zn+2
]. A similar strategy for alkylation of thiol groups is employed in protein farnesyltransferase [5
], geranylgeranyltransferase [6
], methanol:CoM methyltransferase (MtaA) [7
], the E. coli
DNA repair Ada protein [8
], and betaine–Hcy methyltransferase (BHMT) [9
]. However, the sets of zinc ligands and the structures that house the zinc-binding sites are not conserved within this functional family. In particular, the metal ligands and their positions in the sequence are not the same in MetH and MetE. Three cysteines bind the essential zinc in MetH; the first cysteine ligand resides at the end of strand 6 of a (βα)8
barrel, and the remaining vicinal cysteine ligands follow strand 8. A histidine and two cysteines have been identified as metal ligands in E. coli
MetE by a combination of mutagenesis experiments [10
] and extended X-ray absorption fine structure (EXAFS) measurements [12
]. The relative positions of these residues in the sequence led to the prediction that in a (βα)8
MetE barrel the histidine and cysteine ligands would reside at the ends of strands 5 and 8 [4
In contrast, the sequences of MetE enzymes give few if any clues to the strategy for binding and activation of folate by MetE. Thus, the mode of folate binding is a key question to be addressed by structure analysis. In both MetE and MetH, activation of the leaving group is thought to involve the protonation of CH3
folate in a ternary complex, E·Hcy·CH3
folate in MetE, or E·cob(I)alamin·CH3
folate in MetH [4
]. However, the residues that may facilitate protonation have not been identified for either enzyme.
MetE appears to have evolved through gene duplication of a sequence encoding a domain of approximately 340 residues that binds and activates Hcy. Within the family of MetE enzymes (), the N- and C-terminal halves exhibit significant sequence homology. The C-terminal half is more highly conserved than the N-terminal half and has homologs in archae and elsewhere. Among these thiol methyltransferases are several enzymes that are approximately half the size of MetE and utilize corrinoid proteins, rather than folates, as methyl donors. Taken together, these observations suggested that the MetE gene arose as the result of a primordial gene duplication event followed by loss of zinc- and Hcy-binding determinants from the duplicated sequence [11
]. If this hypothesis is correct, the two halves of the MetE sequence should display structural homology, and the N-terminal domain should be more closely related to the C-terminal domain than to any other protein in the database.
Multiple Alignment of MetE from T. maritima (METE_THEMA), E. coli (METE_ECOLI), Saccharomyces cerevisiae (METE_YEAST), and A. thaliana (METE_ARATH)
To determine how MetE has assembled an active site for catalysis of direct methyl transfer from CH3-H4folate to Hcy, we have solved the crystal structure of Thermotoga maritima MetE at 2.0 Å resolution, along with structures of the binary substrate complexes with Hcy and folate. Difficulties in crystallization of the E. coli enzyme were circumvented by analyzing the MetE from T. maritima. This thermophilic bacterium encodes orthologs of E. coli MetH and E. coli MetE. T. maritima MetE (TM1286) is 41% identical to the E. coli enzyme and is only 19 residues shorter than E. coli MetE (), making it an excellent prototype for the MetE family. MetE comprises two (βα)8 barrels. To our knowledge, it is the first example of a dual-(βα)8 barrel enzyme in which the active site is located between barrels arranged in a head-to-head orientation. MetE also provides a rare example of a catalytic zinc site in which four residues serve as metal ligands. Repetition of features within the structure supports the idea that MetE evolved through gene duplication of a primordial zinc/Hcy (βα)8 barrel.