Conformational dynamics associated with remote residues regulate the kinetic properties of homologous glutamate dehydrogenases (GDHs)

Glutamate dehydrogenase (GDH) is a key 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. The crystal structures and first-ever cryo-EM structures of apo-AtGDH captured arrays of conformational ensembles. Comparative structural analysis has revealed a wider mouth opening in allosteric AnGDH ([~] 21 [A]) compared to non-allosteric AtGDH ([~]17 [A]) in their apo states. A network of interaction related to the amino acid substitutions in Domain II is responsible for differential structural dynamics in these GDHs. Remarkably, we identified one remotely located substitution in Domain II, i.e., R246 to S, a part of the network, which reversed the kinetic properties of AtGDH into an allosteric one and controls the mouth opening. Our data also indicate that dynamic discrepancy influences the substrate binding affinity and catalytic activity in AnGDH and AtGDH. We have successfully demonstrated for the first time, that remotely located residues and the conformational dynamics regulate the kinetic properties in homologous GDHs.