Abstact

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 cryo-EM 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. Cryo-EM 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.