Molecular insights into the inhibition of glutamate dehydrogenase by the dicarboxylic acid metabolites

Abstract

Glutamate dehydrogenase (GDH) is a salient metabolic enzyme which catalyzes the NAD + -or NADP + -dependent reversible conversion of α-ketoglutarate (AKG) to L-glutamate; and thereby connects the carbon and nitrogen metabolism cycles in all living organisms. The function of GDH is extensively regulated by both metabolites (citrate, succinate, etc.) and non-metabolites (ATP, NADH, etc.) but sufficient molecular evidences are lacking to rationalize the inhibitory effects by the metabolites. We have expressed and purified NADP + -dependent Aspergillus terreus GDH (AtGDH) in recombinant form. Succinate, malonate, maleate, fumarate, and tartrate independently inhibit the activity of AtGDH to different extents. The crystal structures of AtGDH complexed with the dicarboxylic acid metabolites and the coenzyme NADPH have been determined. Although AtGDH structures are not complexed with substrate; surprisingly, they acquire super closed conformation like previously reported for substrate and coenzyme bound catalytically competent Aspergillus niger GDH (AnGDH). These dicarboxylic acid metabolites partially occupy the same binding pocket as substrate; but interact with varying polar interactions and the coenzyme NADPH binds to the Domain-II of AtGDH. The low inhibition potential of tartrate as compared to other dicarboxylic acid metabolites is due to its weaker interactions of carboxylate groups with AtGDH. Our results suggest that the length of carbon skeleton and positioning of the carboxylate groups of inhibitors between two conserved lysine residues at the GDH active site might be the determinants of their inhibitory potency. Molecular details on the dicarboxylic acid metabolites bound AtGDH active site architecture presented here would be applicable to GDHs in general.

Structural Basis for the Binding of Allosteric Activators Leucine and ADP to Mammalian Glutamate Dehydrogenase

“…The structures show the open form of the enzyme, where the GDH active sites are easily accessible to the substrates, under significantly increased (down to 2.4 Å) resolution, compared to the previously known structures of the GDH open form with ADP (3.5 Å). While the structures of bacterial or fungal GDHs have been deposited at resolutions down to 1.7 Å (PDB 5GUD and 7ECR) [ 17 , 18 ], the lower resolution of mammalian GDH in X-ray crystallography correlates with the presence of a 50-residue “antenna” domain, proposed to be an evolutionary acquisition for interactive regulation of the GDH activity by its multiple allosteric ligands [ 9 ]. Although single particle cryo-EM has been used to overcome the limitation, cryo-EM complexes of mammalian GDH with either GTP or NADH have only been reported at a medium resolution (3.3 Å) [ 10 ].…”

Structural Basis for the Binding of Allosteric Activators Leucine and ADP to Mammalian Glutamate Dehydrogenase

“…We report here new structures of mammalian GDH complexes with the allosteric activators ADP and leucine, which show the open form of the enzyme under significantly increased (down to 2.4 Å) resolution, compared to the previously known structures of the GDH open form with ADP (3.5 Å). While structures of bacterial or fungal GDHs have been deposited at resolutions down to 1.7 Å (PDB 5GUD and 7ECR) [16,17], the lower resolution of mammalian GDH in X-ray crystallography seems to correlate with the presence of a 50-residue "antenna" domain, participating in allosteric regulation. Although single particle cryo-electron microscopy (cryo-EM) has been used to overcome these limitations and provided bovine GDH models at resolutions down to 1.8 Å (PDB 5K12, which is however missing most of the NAD(P)(H) binding domains and significant parts of the N-and C-terminal ends [18]), the complexes of mammalian GDH with either GTP or NADH solved so far by cryo-EM have not yet contributed significant new insights, in part because of their resolutions still not exceeding 3.3 Å [12].…”

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

“…The expression plasmid construct confirmed by sequencing was then transformed into Δ gdh E. coli BL21 (DE3) cells. For AtGDH, the construct mentioned previously (Godsora et al, 2022) was used.…”

“…Recombinant WT AtGDH was expressed, and Ni-NTA affinity and size exclusion chromatography were used to purify the enzyme as described previously (Godsora et al, 2022). The cell containing the plasmid bearing Angdh construct was inoculated in 5 ml LB broth containing kanamycin (50 µg/ml) overnight.…”

“…The NADP + -dependent GDH catalyzes the reductive amination of α-ketoglutarate to L-glutamate in the presence of excess ammonia and coenzyme NADPH, termed forward activity. The forward activity of AtGDH and AnGDH was measured using the method described earlier (Noor & Punekar, 2005; Prakash et al, 2018; Agarwal et al, 2019; Godsora et al, 2022).…”