Conformational flexibility associated with remote residues regulates the kinetic properties of glutamate dehydrogenase.

Glutamate dehydrogenase (GDH) is a pivotal metabolic enzyme in all living organisms, and some of the GDHs exhibit substrate-dependent homotropic cooperativity. However, the mode of allosteric communication during the homotropic effect in GDHs remains poorly understood. In this study, we examined two homologous GDHs, Aspergillus niger GDH (AnGDH) and Aspergillus terreus GDH (AtGDH), with differing substrate utilization kinetics to uncover the factors driving their distinct behavior. We report the crystal structures and first-ever cryo-EM structures of apo- AtGDH and AnGDH that captured arrays of conformational ensembles. A wider mouth opening (~ 21 Å) is observed for the cooperative AnGDH as compared to the non-cooperative AtGDH (~17 Å) in their apo states. A network of interactions related to the substitutions in Domain II influence structural flexibility in these GDHs. Remarkably, we have identified a distant substitution (R246 to S) in Domain II, as a part of this network, which can impact the mouth opening and converts non-cooperative AtGDH into a cooperative enzyme. Our study demonstrates that remote residues can influence structural and kinetic properties in homologous GDHs.