Structural Snapshots of B12-Dependent Methionine Synthase's Catalytic Conformations

Abstract

Cobalamin (vitamin B12) and its derivatives play an essential role in biological methylation, with cobalamin-dependent methionine synthase (MS) serving as a canonical example. MS catalyzes multiple methyl transfers within a single, dynamic multi-domain architecture that has proven challenging to study, hampering efforts to elucidate its catalytic mechanism(s). Utilizing a thermostable MS homolog and non-native cobalamin cofactors, we have captured crystal structures of transient conformational states of MS, including those directly involved in folate demethylation and homocysteine methylation. These snapshots reveal the mechanistic significance of five-coordinate, His-off methylcobalamin in homocysteine methylation and highlight the crucial role of the folate-binding domain and interdomain linkers in orchestrating the intricate structural rearrangements required for catalysis. This expanded conformational ensemble, including the unexpected capture of novel Cap-on conformations, underscores the remarkable plasticity of MS, exceeding previous estimations. Our findings provide crucial insights into the catalytic mechanism of MS, laying the foundation for harnessing cobalamins biocatalytic potential and elucidating how nature exploits protein dynamics to facilitate complex transformations.