Tertiary and quaternary structure remodeling by occupancy of the substrate binding pocket in a large glutamate dehydrogenase

Glutamate dehydrogenases (GDHs) catalyze the oxidative deamination of L-glutamate to 2-oxoglutarate using NAD(P) ⁺ as a cofactor. The large type of GDHs (L-GDHs) displays a dynamic homotetrameric architecture that alternates between open and closed states. However, the catalytic mechanism and the functional relevance of the large conformational changes in L-GDHs remain poorly understood. Here, we use cryoEM to investigate the structure and the conformational landscape of the mycobacterial L-GDH composed of 180 kDa subunits (mL-GDH ₁₈₀ ) when incubated with L-glutamate and NAD ⁺ . Classification of the heterogeneous population of tetramers reveals opening-closing motions and sorting of individual subunits resolves the occupancy of the cofactor and substrate binding pockets. CryoEM maps show that ligand binding to the glutamate binding pocket is accompanied by structural changes in a region approximately two nanometers away from the active site, leading to the formation of a previously undetected interaction between the catalytic domains of neighboring subunits in mL-GDH ₁₈₀ closed tetrameric states. Our findings indicate that the occupancy of the substrate binding site of mL-GDH ₁₈₀ is linked to a remodeling of both the tertiary and quaternary structure of the enzyme.