S-Adenosylmethionine synthetase (MAT) catalyzes formation of S-adenosylmethionine (SAM) from ATP and l-methionine (Met) and hydrolysis of tripolyphosphate to PP(i) and P(i). Escherichia coli MAT (eMAT) has been crystallized with the ATP analogue AMPPNP and Met, and the crystal structure has been determined at 2.5 A resolution. eMAT is a dimer of dimers and has a 222 symmetry. Each active site contains the products SAM and PPNP. A modeling study indicates that the substrates (AMPPNP and Met) can bind at the same sites as the products, and only a small conformation change of the ribose ring is needed for conversion of the substrates to the products. On the basis of the ternary complex structure and a modeling study, a novel catalytic mechanism of SAM formation is proposed. In the mechanism, neutral His14 acts as an acid to cleave the C5'-O5' bond of ATP while simultaneously a change in the ribose ring conformation from C4'-exo to C3'-endo occurs, and the S of Met makes a nucleophilic attack on the C5' to form SAM. All essential amino acid residues for substrate binding found in eMAT are conserved in the rat liver enzyme, indicating that the bacterial and mammalian enzymes have the same catalytic mechanism. However, a catalytic mechanism proposed recently by González et al. based on the structures of three ternary complexes of rat liver MAT [González, B., Pajares, M. A., Hermoso, J. A., Guillerm, D., Guillerm, G., and Sanz-Aparicio. J. (2003) J. Mol. Biol. 331, 407] is substantially different from our mechanism.

The crystal structure of rat liver S-adenosyl-L-homocysteine hydrolase (AdoHcyase, EC 3.3.1.1) which catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) has been determined at 2.8 A resolution. AdoHcyase from rat liver is a tetrameric enzyme with 431 amino acid residues in each identical subunit. The subunit is composed of the catalytic domain, the NAD+-binding domain, and the small C-terminal domain. Both catalytic and NAD+-binding domains are folded into an ellipsoid with a typical alpha/beta twisted open sheet structure. The C-terminal section is far from the main body of the subunit and extends into the opposite subunit. An NAD+ molecule binds to the consensus NAD+-binding cleft of the NAD+-binding domain. The peptide folding pattern of the catalytic domain is quite similar to the patterns observed in many methyltransferases. Although the crystal structure does not contain AdoHcy or its analogue, there is a well-formed AdoHcy-binding crevice in the catalytic domain. Without introducing any major structural changes, an AdoHcy molecule can be placed in the catalytic domain. In the structure described here, the catalytic and NAD+-binding domains are quite far apart from each other. Thus, the enzyme appears to have an "open" conformation in the absence of substrate. It is likely that binding of AdoHcy induces a large conformational change so as to place the ribose moiety of AdoHcy in close proximity to the nicotinamide moiety of NAD+. A catalytic mechanism of AdoHcyase has been proposed on the basis of this crystal structure. Glu155 acts as a proton acceptor from the O3'-H when the proton of C3'-H is abstracted by NAD+. His54 or Asp130 acts as a general acid-base catalyst, while Cys194 modulates the oxidation state of the bound NAD+. The polypeptide folding pattern of the catalytic domain suggests that AdoHcy molecules can travel freely to and from AdoHcyase and methyltransferases to properly regulate methyltransferase activities. We believe that the crystal structure described here can provide insight into the molecular architecture of this important regulatory enzyme.

Methyltransfer reactions are some of the most important reactions in biological systems. Glycine N-methyltransferase (GNMT) catalyzes the S-adenosyl-l-methionine- (SAM-) dependent methylation of glycine to form sarcosine. Unlike most SAM-dependent methyltransferases, GNMT has a relatively high value and is weakly inhibited by the product S-adenosyl-l-homocysteine (SAH). The major role of GNMT is believed to be the regulation of the cellular SAM/SAH ratio, which is thought to play a key role in SAM-dependent methyltransfer reactions. Crystal structures of GNMT complexed with SAM and acetate (a potent competitive inhibitor of Gly) and the R175K mutated enzyme complexed with SAM were determined at 2.8 and 3.0 A resolutions, respectively. With these crystal structures and the previously determined structures of substrate-free enzyme, a catalytic mechanism has been proposed. Structural changes occur in the transitions from the substrate-free to the binary complex and from the binary to the ternary complex. In the ternary complex stage, an alpha-helix in the N-terminus undergoes a major conformational change. As a result, the bound SAM is firmly connected to protein and a "Gly pocket" is created near the bound SAM. The second substrate Gly binds to Arg175 and is brought into the Gly pocket. Five hydrogen bonds connect the Gly in the proximity of the bound SAM and orient the lone pair orbital on the amino nitrogen (N) of Gly toward the donor methyl group (C(E)) of SAM. Thermal motion of the enzyme leads to a collision of the N and C(E) so that a S(N)2 methyltransfer reaction occurs. The proposed mechanism is supported by mutagenesis studies.