Making Methionine

Abstract

Biomolecules are fundamental to life, and their structures dictate their functions. Cofactors are essential for expanding the catalytic capabilities of enzymes, enabling them to perform a diverse range of chemical transformations. While structural biology has advanced significantly and rapidly within the past decade, capturing the dynamism inherent to many biomolecules remains a fundamental challenge. This work aims to bridge this gap by investigating the dynamic interplay between structure, cofactors, and function in two key enzymes involved in one-carbon metabolism: methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MS). These enzymes exhibit remarkable conformational flexibility, enabling them to catalyze complex transformations. MTHFR, a flavin-dependent enzyme, plays a crucial role in folate metabolism and is subject to allosteric regulation by S-adenosylmethionine (SAM). Its active site exhibits remarkable plasticity to accommodate chemically distinct substrates and carry out redox chemistry and C-N bond/ring cleavage. MS, a cobalamin-dependent enzyme, catalyzes multiple methyl transfers essential for methionine synthesis. MS displays remarkable structural flexibility to foster three chemically distinct methylations, using methyltetrahydrofolate (MTF), a tertiary amine, as a methyl donor and shuttling this group to homocysteine (HCY), an unreactive thiol, at physiological pH, acting as a scavenger for the proteogenic amino acid methionine (MET). Despite their importance, a comprehensive understanding of the molecular mechanisms underpinning these enzymes has been hindered by their dynamic nature and the lack of complete structural information, a consequence of the lack of a viable structural-functional model. To overcome this challenge, we employed a multidisciplinary approach combining synthetic biology, biochemistry, and structural biology, utilizing thermostable homologs of these enzymes. This strategy allowed us to obtain the first high-resolution structure of full-length MS, visualize cobalamin loading, and capture transient catalytic states, providing the first structural blueprints of a cobalamin-dependent enzyme captured in action (folate demethylation, homocysteine methylation). For MTHFR, we captured both active and inactive conformations, revealing a unique allosteric mechanism coupled with dual SAM binding, providing the first structural and functional MTHFR model towards understanding how Nature makes use of unstructured regions to achieve allostery and orchestrate dynamic rearrangements. To the best of our knowledge, this linker-mediated allostery is unprecedented and represents a mode of allosteric regulation that grants significance to traditionally overlooked linker regions. These findings provide crucial insights into how these enzymes utilize cofactors and protein dynamics to perform improbable chemistries. This knowledge forms the basis for developing new biocatalysts with tailored activities. Moreover, these structural blueprints can be used to map disease-associated polymorphisms, ultimately leading to a deeper understanding of disease mechanisms and the development of novel therapeutic strategies. By providing structural blueprints of these dynamic enzymes in action, this work paves the way for future applications in biocatalysis and drug discovery.