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

Abstract

Glutamate dehydrogenase (GDH) plays a key role in the metabolism of glutamate, an important compound at a cross-road of carbon and nitrogen metabolism and a relevant neurotransmitter. Despite being one of the first discovered allosteric enzymes, GDH still poses challenges for structural characterization of its allosteric sites. Only the structures with ADP, and at low (3.5 Å) resolution, are available for mammalian GDH complexes with allosteric activators. Here, we aim at deciphering a structural basis for the GDH allosteric activation using bovine GDH as a model. For the first time, we report a mammalian GDH structure in a ternary complex with the activators leucine and ADP, co-crystallized with potassium ion, resolved to 2.45 Å. An improved 2.4-angstrom resolution of the GDH complex with ADP is also presented. The ternary complex with leucine and ADP differs from the binary complex with ADP by the conformation of GDH C-terminus, involved in the leucine binding and subunit interactions. The potassium site, identified in this work, may mediate interactions between the leucine and ADP binding sites. Our data provide novel insights into the mechanisms of GDH activation by leucine and ADP, linked to the enzyme regulation by (de)acetylation.

The Mitochondrial Protein MitoNEET as a Probe for the Allostery of Glutamate Dehydrogenase

“…A recent publication allowed the examination of the structure of the ternary complex of bovine GDH-ADP-leucine for the first time (PDB structure 8AR7) [ 23 ]. Additionally, a potassium ion was co-crystallized in the structure and hypothesized to be an additional ligand.…”

Evolutionary Changes in Primate Glutamate Dehydrogenases 1 and 2 Influence the Protein Regulation by Ligands, Targeting and Posttranslational Modifications

“…Additionally, leucine [22], diethylstilbestrol/estrogens [9], palmitoyl-CoA [23] and Zn 2+ [24] have also been known as the natural regulators of the mammalian GDH. Of these natural regulators, localization of a Zn 2+ binding site [25] and, only recently, a leucine binding site found together with a novel K + ion site [10] have been identified (Figure 1). Surprisingly, all these ligands possess separate allosteric regulatory sites, and even more binding sites have been identified with the help of crystallization screenings.…”

“…Thus, eight different ligand binding sites other than the active site are available in mammalian GDH (Figure 1). However, despite more than 20 structures of mammalian GDH available in PDB, the binding sites of even natural regulators such as palmitoyl-CoA [23], estrogens/diethylstilbestrol [9,28,29], thiamine or its deriva-tives [10,30], or haloperidol, perphenazine [29] and many other ligands [31,32] remain to be identified.…”

“…Mammalian GDH was one of the first discovered allosteric enzymes [9]. Still, the available structural data do not allow complete characterization of GDH regulation by its ligands, and the accumulation of new data fills the gap, providing further understanding of GDH allosteric regulation [10].…”

Mitolnc controls cardiac BCAA metabolism and heart hypertrophy by allosteric activation of BCKDH

“…Alternatively, leucine concentration might also be detected by the leucyl tRNA synthetase 1 (LARS1) ( 16 ), whose activity is glucose-dependent ( 17 ), illustrating the complexity of regulatory networks involving leucine. Another important signaling effect of leucine is the allosteric activation of glutamate dehydrogenase (GDH) ( 18 , 19 ), yielding α-ketoglutarate.…”