The Cobalamin-Independent Methionine Synthase Enzyme Captured in a Substrate-Induced Closed Conformation

Abstract

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 (βα)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.

Fungal cobalamin-independent methionine synthase: Insights from the model organism, Neurospora crassa

“…Finally, structural analysis and site directed mutagenesis indicate that His 705 would act as a key catalytic acid, protonating the folate substrate through a water intermediate [29]. As expected for such a role, this residue is universally conserved in the sequences analyzed.…”

“…As expected for such a role, this residue is universally conserved in the sequences analyzed. The side chain of this residue is undefined in many C. albicans structures, but defined in the closed conformation structure [29].…”

“…In examining the domain closure of MetE, it was found that both domains act as rigid bodies, and closure involves a rotation around an axis that is slightly off parallel from a plane through the central cleft [29]. The residues that are in contact between the domains are identified in As this interaction does not involve the side chain, there is no requirement for Val 709 to be conserved, and the sequence analysis indicated that it was not.…”

“…Interest has focused on the Zn 2+ ion positioned at the active site, which seems to have a role in activating the homocysteine substrate. Interestingly, this zinc ion is found to be mobile and to move towards homocysteine upon substrate binding [27][28][29].…”

“…Initial structures from A. thaliana [9] and T. maritima [7] placed the folate methyl group greater than 7 Å from the homocysteine sulfur, too distant to constitute a catalytically productive complex. Recently, a C. albicans structure was reported with the substrate, homocysteine, and the product, H 4 PteGlu 3, brought in close proximity by a conformational change [29]. While site directed mutations suggested that the folate binding site was correctly identified, this structure (PDB 4QQU) actually places the two ligands in rather tight proximity, such that there would be insufficient space to accommodate a reactive methyl group.…”