Orchestrating Improbable Chemistries: Structural Snapshots of B12-Dependent Methionine Synthase's Catalytic Choreography

Cobalamin (vitamin B₁₂) and its derivatives are used to power chemical transformations crucial for life. Among these essential reactivities are methylations, of which cobalamin-dependent methionine synthase (MS) is the canonical example. MS catalyzes three distinct methyl transfers central to one-carbon metabolism. Despite its importance in the biological methyl cycle and relevance to human health, fundamental studies on the molecular basis of MS catalysis have proven elusive due to substantial biochemical challenges associated with MS from traditional sources. Here, we leverage our previously established thermophilic model system (tMS), its remarkable stability, and its ability to bind non-native cobalamin cofactors to systematically capture previously unattainable conformations via crystallography, expanding the conformational ensemble of MS to include gating conformations and present the first structures of a cobalamin enzyme in action (folate demethylation and homocysteine methylation). We establish the role of the folate (Fol) domain and its associated linkers in triggering the structural transitions required for activity. Our work highlights the importance of linkers in mediating large-scale rearrangements that underpin the catalysis of improbable chemistries. By providing the first structural blueprints associated with two cobalamin-mediated methyl transfers, we lay the groundwork for exploring cobalamin's biocatalytic potential and provide a framework by which Nature harnesses dynamic motions to achieve dynamic chemical outcomes.