CRYO-EM STUDIES ON CONFORMATIONAL CHANGES IN COBALAMIN-DEPENDENT MUTASES AND METAL-INDUCED TAU

Adenosylcobalamin (AdoCbl)-dependent mutases are essential for radical-mediated rearrangements in amino acid metabolism and microbial fermentation, initiating catalysis through homolytic cleavage of the Co(III)-C bond in AdoCbl to generate a reactive 5′-deoxyadenosyl radical. Despite their importance, the structural mechanisms governing radical generation and cofactor stabilization remain poorly understood. This dissertation employs cryo-electron microscopy (cryo-EM) to elucidate the conformational dynamics underlying radical initiation in two model systems: lysine 5,6-aminomutase (5,6-LAM) and ornithine 4,5-aminomutase (4,5-OAM). High-resolution cryo-EM structures of these enzymes in substrate-free and substrate-bound states reveal extensive domain rearrangements that reposition AdoCbl and the pyridoxal phosphate (PLP) cofactor, enabling catalytic activation. In 5,6-LAM, substrate binding induces global domain closure, weakening the Co(III)-C bond and priming the enzyme for radical formation. In 4,5-OAM, substrate engagement triggers large-scale conformational changes that enhance active site organization, with the accessory subunit OraS playing a critical role in stabilizing the closed state. Electron paramagnetic resonance (EPR) spectroscopy further confirms the presence of transient radical intermediates, validating the proposed activation mechanism. These findings provide a structural framework for understanding AdoCbl-dependent catalysis, highlighting the role of domain motions in enzyme activation. By integrating structural and mechanistic insights, this work advances our understanding of radical enzymology and lays the foundation for future enzyme engineering efforts aimed at optimizing biocatalysis and metabolic pathways.