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. The catalytic mechanism and the role of the large conformational changes of L-GDHs in enzymatic function are unknown. Here, we explore by cryoEM the structure and the conformational space 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 into different subsets reveals opening-closing motions and sorting of individual subunits resolves the occupancy of the cofactor and substrate binding pockets. Maps show that ligand binding to the glutamate binding pocket is accompanied by structural changes in a distant region that mediates a novel interaction between the catalytic domains of neighboring subunits in closed tetrameric states. Our findings indicate that the occupancy of the orthosteric site of mL-GDH ₁₈₀ is linked to a remodeling of the enzyme tertiary and quaternary structure.