The cobalamin-independent methionine synthase enzyme captured in a substrate-induced closed conformation.

The cobalamin-independent methionine synthase enzyme catalyzes a challenging reaction: the direct transfer of a methyl from 5-methyl-tetrahydrofolate-glutamate3 to the l-homocysteine thiol. The enzyme has a dual (ßα)8 TIM barrel structure that binds, activates and brings the reactants into reaction proximity by conformational movements. In the previously observed open structures, the substrates bind too far apart to react, but we have captured a ternary complex with both substrates bound in a closed form of the enzyme. The closing is described in terms of a hinge between the N- and C-terminal TIM barrels and a rearrangement of key loops within the C domain. The substrate specificity can now be rationalized and the structure reveals His707 as the acid that protonates the THF leaving group through a water molecule trapped in the closed active site. The substrates are correctly oriented for an in-line attack by l-homocysteine on the N(5)-methyl.

Structural analysis of a fungal methionine synthase with substrates and inhibitors.

The cobalamin-independent methionine synthase from Candida albicans, known as Met6p, is a 90-kDa enzyme that consists of two (ßα)8 barrels. The active site is located between the two domains and has binding sites for a zinc ion and substrates L-homocysteine and 5-methyl-tetrahydrofolate-glutamate3. Met6p catalyzes transfer of the methyl group of 5-methyl-tetrahydrofolate-glutamate3 to the L-homocysteine thiolate to generate methionine. Met6p is essential for fungal growth, and we currently pursue it as an antifungal drug design target. Here we report the binding of L-homocysteine, methionine, and several folate analogs. We show that binding of L-homocysteine or methionine results in conformational rearrangements at the amino acid binding pocket, moving the catalytic zinc into position to activate the thiol group. We also map the folate binding pocket and identify specific binding residues, like Asn126, whose mutation eliminates catalytic activity. We also report the development of a robust fluorescence-based activity assay suitable for high-throughput screening. We use this assay and an X-ray structure to characterize methotrexate as a weak inhibitor of fungal Met6p.

Structure of Candida albicans methionine synthase determined by employing surface residue mutagenesis.

Fungal methionine synthase, Met6p, transfers a methyl group from 5-methyl-tetrahydrofolate to homocysteine to generate methionine. The enzyme is essential to fungal growth and is a potential anti-fungal drug design target. We have characterized the enzyme from the pathogen Candida albicans but were unable to crystallize it in native form. We converted Lys103, Lys104, and Glu107 all to Tyr (Met6pY), Thr (Met6pT) and Ala (Met6pA). All variants showed wild-type kinetic activity and formed useful crystals, each with unique crystal packing. In each case the mutated residues participated in beneficial crystal contacts. We have solved the three structures at 2.0-2.8Å resolution and analyzed crystal packing, active-site residues, and similarity to other known methionine synthase structures. C. albicans Met6p has a two domain structure with each of the domains having a (ßα)(8)-barrel fold. The barrels are arranged face-to-face and the active site is located in a cleft between the two domains. Met6p utilizes a zinc ion for catalysis that is bound in the C-terminal domain and ligated by four conserved residues: His657, Cys659, Glu679 and Cys739.