Structural analysis and discovery of lead compounds for the fungal methionine synthase enzyme

Methionine synthases catalyze methyl transfer from 5-methyl-tetrahydrofolate (5-methyl-THF) to L-homocysteine (Hcy) in order to generate methionine (Met). Mammals, including humans, use a cobalamin dependent form, while fungi use a cobalamin independent protein called Met6p. The large structural differences between them make Met6p a potential anti-fungal drug target. Met6p is a 90 kDa protein with the active site located between two (βα)₈ barrels. The active site has a catalytic Zn²+ and binding sites for the two substrates, Hcy and folate. I present the crystal structures of three engineered variants of the Met6p enzyme from Candida albicans. I also solved Met6p in complex with several substrate and product analogs, including Hcy, Met, Gln, 5-methyl-THF-Glu₃ and Methotrexate-Glu₃ (MTX-Glu₃), and the bi-dentate ligand S-adenosyl homocysteine. Also described is a new fluorescence-based activity assay monitoring Hcy. Lastly, a high-throughput Differential Scanning Fluorimetry (DSF) assay was used to screen thousands of compounds in order to identify ligands which bind Met6p. My work details the mode of interaction of Hcy and folate with the Met6p protein. Several residues important to activity were discovered, like Asn 126 and Tyr 660, and proven to be important by site directed mutagenesis. Structural analysis revealed an important aspect of the mechanism. When Hcy binds to its pocket it makes strong ion pairs with the enzyme. In particular, 614 moves toward the substrate amine and triggers a rearrangement of active site loops; this draws the catalytic Zn²+ toward the Hcy thiol where a new ligand bond is formed, activating the thiol for methyl transfer. The work presented here lays the groundwork for structure based drug design and makes the development of Met6p specific bi-dentate ligands feasible. The fluorescence based activity assay I developed was successfully used to test the folate analog MTX-Glu₃, which inhibits with an IC₅₀ of ~4 mM. I also discovered our first bi-dentate ligand in the form of S-adenosyl homocysteine.