Crystal structure of glutamate dehydrogenase 2, a positively selected novel human enzyme involved in brain biology and cancer pathophysiology

Abstract

Introduction: Mammalian glutamate dehydrogenase (hGDH1 in human cells) interconverts glutamate to α-ketoglutarate and ammonia while reducing NAD(P) to NAD(P)H. During primate evolution, humans and great apes have acquired hGDH2, an isoenzyme that underwent rapid evolutionary adaptation concomitantly with brain expansion, thereby acquiring unique catalytic and regulatory properties that permitted its function under conditions inhibitory to its ancestor hGDH1. Although the 3D-structures of GDHs, including hGDH1, have been determined, attempts to determine the hGDH2 structure were until recently unsuccessful. Comparison of the hGDH1/hGDH2 structures would enable a detailed understanding of their evolutionary differences. This work aimed at the determination of the hGDH2 crystal structure and the analysis of its functional implications. Recombinant hGDH2 was produced in the Spodoptera frugiperda ovarian cell line Sf21, using the Baculovirus expression system. Purification was achieved via a two-step chromatography procedure. hGDH2 was crystallized, X-ray diffraction data were collected using synchrotron radiation and the structure was determined by molecular replacement. The hGDH2 structure is reported at a resolution of 2.9 Å. The enzyme adopts a novel semi-closed conformation, which is an intermediate between known open and closed GDH1 conformations, differing from both. The structure enabled us to dissect previously reported biochemical findings and to structurally interpret the effects of evolutionary amino acid substitutions, including Arg470His, on ADP affinity. In conclusion, our data provide insights into the structural basis of hGDH2 properties, the functional evolution of hGDH isoenzymes, and open new prospects for drug design, especially for cancer therapeutics. | 803 DIMOVASILI et AL.

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

“…The mammalian GDH K + binding site described here is structurally equivalent to the K + site identified in GDH1 from Arabidopsis thaliana (6YEH; [20]), and to one of the multiple Na + -binding sites in the structure of human GDH2 (6G2U; [21]) (Figure 4). Among bacterial GDH with the resolved structures, only GDH from Corynebacterium glutamicum contains multiple sites for potassium ion binding (5GUD; [16]), one of them coinciding with the K + site of mammalian GDH identified by us.…”

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

“…The K + binding site of mammalian GDH, that is described here, is structurally equivalent to the K + site identified in GDH1 from Arabidopsis thaliana (6YEH [ 21 ]) and to one of the multiple Na + binding sites in the structure of human GDH2 (6G2U [ 22 ]) ( Figure 4 ). Among bacterial GDH with the resolved structures, only GDH from Corynebacterium glutamicum contains multiple sites for potassium ion binding (5GUD [ 17 ]), one of them coinciding with the K + site of mammalian GDH identified by us.…”

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

“…AtGDH and AnGDH were purified as recombinant enzymes, and the structures were determined using X-ray crystallography and cryo-EM to understand the molecular switch that regulates the kinetic properties of these enzymes. AnGDH and AtGDH are hexameric enzymes with a canonical tertiary structural fold like other hexameric GDHs (Baker et al, 1992; Werner et al, 2005; Dimovasili et al, 2021). Only one previous report presented the structural evidences using X-ray crystallography and MD simulation indicating the presence of heterogeneous opening and closing of the active site in the apo form of a Thermococcus profundus GDH (TpGDH) (PDB ID: 1EUZ) (Oroguchi & Nakasako, 2016).…”