Martha L. Ludwig

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. coli MTHFR, the A → V mutation increases the rate of dissociation of FAD, and in both enzymes, loss of FAD is linked to changes in quaternary structure [Yamada, K., Chen, Z., Rozen, R., and Matthews, R. G. (2001) Proc. Natl. Acad. Sci.U.S.A.98, 14853−14858; Guenther, B. D., Sheppard, C. A., Tran, P., Rozen, R., Matthews, R. G., and Ludwig, M. L. (1999) Nat. Struct. Biol.6, 359−365]. Folates have been shown to protect both human and bacterial enzymes from loss of FAD. Despite its effect on affinity for FAD, the A → V mutation is located at the bottom of the (βα)₈ barrel of the catalytic domain in a position that does not contact the bound FAD prosthetic group. Here we report the structures of the Ala177Val mutant of E. coli MTHFR and of its complex with the 5,10-dideazafolate analogue, LY309887, and suggest mechanisms by which the mutation may perturb FAD binding. Helix α5, which immediately precedes the loop bearing the mutation, carries several residues that interact with FAD, including Asn168, Arg171, and Lys172. In the structures of the mutant enzyme this helix is displaced, perturbing protein−FAD interactions. In the complex with LY309887, the pterin-like ring of the analogue stacks against the si face of the flavin and is secured by hydrogen bonds to residues Gln183 and Asp120 that adjoin this face. The direct interactions of bound folate with the cofactor provide one mechanism for linkage between binding of FAD and folate binding that could account in part for the protective action of folates. Conformation changes induced by folate binding may also suppress dissociation of FAD.

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 (βα)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)₃Zn 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.

B ₁₂ -dependent methionine synthase (MetH) is a large modular enzyme that utilizes the cobalamin cofactor as a methyl donor or acceptor in three separate reactions. Each methyl transfer occurs at a different substrate-binding domain and requires a different arrangement of modules. In the catalytic cycle, the cobalamin-binding domain carries methylcobalamin to the homocysteine (Hcy) domain to form methionine and returns cob(I)alamin to the folate (Fol) domain for remethylation by methyltetrahydrofolate (CH ₃ -H ₄ folate). Here, we describe crystal structures of a fragment of MetH from Thermotoga maritima comprising the domains that bind Hcy and CH ₃ -H ₄ folate. These substrate-binding domains are (βα) ₈ barrels packed tightly against one another with their barrel axes perpendicular. The properties of the domain interface suggest that the two barrels remain associated during catalysis. The Hcy and CH ₃ -H ₄ folate substrates are bound at the C termini of their respective barrels in orientations that position them for reaction with cobalamin, but the two active sites are separated by ≈50 Å. To complete the catalytic cycle, the cobalamin-binding domain must travel back and forth between these distant active sites.

B(12)-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that uses bound cobalamin as an intermediate methyl carrier. Major domain rearrangements have been postulated to explain how cobalamin reacts with three different substrates: homocysteine, methyltetrahydrofolate and S-adenosylmethionine (AdoMet). Here we describe the 3.0 A structure of a 65 kDa C-terminal fragment of MetH that spans the cobalamin- and AdoMet-binding domains, arranged in a conformation suitable for the methyl transfer from AdoMet to cobalamin that occurs during activation. In the conversion to the activation conformation, a helical domain that capped the cofactor moves 26 A and rotates by 63 degrees, allowing formation of a new interface between cobalamin and the AdoMet-binding (activation) domain. Interactions with the MetH activation domain drive the cobalamin away from its binding domain in a way that requires dissociation of the axial cobalt ligand and, thereby, provide a mechanism for control of the distribution of enzyme conformations.