Structural and functional studies of human methionine adenosyltransferases

S-Adenosylmethionine (SAMe) is the principal methyl donor of the cell and is synthesized via an ATP-driven process by methionine adenosyltransferase (MAT) enzymes. It is tightly linked with cell proliferation in liver and colon cancer. In humans, there are three genes, MAT1A, MAT2A and MAT2B, which encode MAT enzymes. MAT2A and MAT2B transcribe MATα2 and MATβ enzyme subunits, respectively, with catalytic and regulatory roles. The MATα2β complex is expressed in nearly all tissues and is thought to be essential in providing the necessary SAMe flux for methylation of DNA and various proteins including histones. In human hepatocellular carcinoma MAT2A and MAT2B genes are upregulated, highlighting the importance of the MATα2β complex in liver disease. The individual subunits have been structurally characterized but the nature of the complex has remained elusive despite its existence having been postulated for more than 20 years and the observation that MATβ is often co-localized with MATα2. Though SAMe can be produced by MAT(α2)4 alone, this thesis shows that the kcat of the MATα2β complex is three- to four fold higher depending on the variants of MAT that participate in complex formation. Using X-ray crystallography and solution Xray scattering, the first structures are provided of this 258 kDa functional complex both in crystals and solution with an unexpected stoichiometry of 4α2 and 2βV2 subunits. It is demonstrated that the N-terminal regulates the activity of the complex and it is shown that complex formation takes place surprisingly via the C-terminal of MATβV2 that buries itself in a tunnel created at the interface of the MAT(α2)2. The structural data suggest a unique mechanism of regulation and provide a gateway for structure-based drug design in anticancer therapies. ii Crystal structures of human MATα2 containing various bound ligands providing a ‘structural movie’ of the catalytic steps are also presented. High to atomic-resolution structures reveal the structural elements of the enzyme involved in the utilization of substrates, methionine and adenosine, and the formation of the product SAMe. MAT enzymes are also able to produce S-adenosylethionine (SAE) from substrate ethionine. Ethionine, an S-ethyl analogue of the amino acid methionine, is known to induce steatosis and pancreatitis. It is shown here that S-adenosylethionine occupies the active site in a manner similar to SAMe, confirming that ethionine also binds and utilizes the same catalytic site to form the product SAE. Through by gel filtration and small angle x-ray scattering (SAXS) it is shown that the catalytic MAT enzymes exist in multiple oligomeric populations in solution, whilst the regulatory MAT subunit, MATβ, is monomeric. In view of these data and recent crystallographic structures of the MAT enzyme complex, a rationalization of nomenclature for the MAT enzymes and their complexes has been produced