Harnessing Large Protein Dynamics to Control Chemical Transformations

Researchers from the University of Michigan are investigating how a vitamin B12-dependent enzyme orchestrates a series of chemically challenging methyl-transfer reactions essential to human metabolism. Methionine synthase (MS) is a large, multi-domain enzyme that plays a central role in one-carbon metabolism, catalyzing reactions between inherently unreactive molecules that would not proceed under normal physiological conditions. A fundamental barrier to understanding this enzyme has been the difficulty of capturing its structure and dynamics during catalysis: MS undergoes extensive domain rearrangements as it cycles through distinct chemical steps, and conventional structural approaches yield only static snapshots. The research team will leverage a thermophilic MS system that enables precise biochemical control, including reconstitution with native and synthetic cobalamin analogs, to trap and characterize the enzyme in its catalytically relevant conformational states. Their discoveries could transform the mechanistic understanding of multi-domain enzyme catalysis and inform the design of new biocatalytic tools relevant to biotechnology and the treatment of methylation-related disorders. The project will also support interdisciplinary training for students and expand the "Science for Tomorrow" outreach program, which delivers hands-on enzyme science to middle school students in the Detroit and Ypsilanti communities. This project will employ an integrated suite of biochemical, bioinorganic, and biophysical methods, including cryo-electron microscopy, X-ray crystallography, spectroscopic approaches, and enzymological assays, to elucidate the molecular mechanisms underlying each of the three MS-catalyzed methylation reactions and the large-scale conformational transitions that support them. A central focus will be characterizing how the oxidation state and coordination environment of the cobalamin cofactor, together with substrate binding, shape the structural ensemble accessible to MS and drive progression through its catalytic cycle. Using the thermophilic MS platform developed in the Koutmos laboratory, the team will systematically introduce mutations, deploy synthetic cobalamin analogs, and trap transient catalytic states to dissect the dynamic interplay between protein structure and chemical function. These studies will build on the first high-resolution, full-length MS structure and the first experimentally captured MS catalytic states, both achieved in this laboratory. The knowledge gained will be applied toward engineering MS-based proteins with altered or expanded substrate scope, establishing a foundation for the design of programmable methyltransferase biocatalysts. Collectively, this work will advance fundamental understanding of cofactor-dependent multi-domain enzymology and open new avenues for enzyme engineering and biotechnological application. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.