The Odyssey of a Young Gene: Structure–Function Studies in Human Glutamate Dehydrogenases Reveal Evolutionary-Acquired Complex Allosteric Regulation Mechanisms

Abstract

Mammalian glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia, interconnecting carbon skeleton and nitrogen metabolism. In addition, it functions as an energy switch by its ability to fuel the Krebs cycle depending on the energy status of the cell. As GDH lies at the intersection of several metabolic pathways, its activity is tightly regulated by several allosteric compounds that are metabolic intermediates. In contrast to other mammals that have a single GDH-encoding gene, humans and great apes possess two isoforms of GDH (hGDH1 and hGDH2, encoded by the GLUD1 and GLUD2 genes, respectively) with distinct regulation pattern, but remarkable sequence similarity (they differ, in their mature form, in only 15 of their 505 amino-acids). The GLUD2 gene is considered a very young gene, emerging from the GLUD1 gene through retro-position only recently (<23 million years ago). The new hGDH2 iso-enzyme, through random mutations and natural selection, is thought to have conferred an evolutionary advantage that helped its persistence through primate evolution. The properties of the two highly homologous human GDHs have been studied using purified recombinant hGDH1 and hGDH2 proteins obtained by expression of the corresponding cDNAs in Sf21 cells. According to these studies, in contrast to hGDH1 that maintains basal activity at 35-40 % of its maximal, hGDH2 displays low basal activity that is highly responsive to activation by rising levels of ADP and/or L-leucine which can also act synergistically. While hGDH1 is inhibited potently by GTP, hGDH2 shows remarkable GTP resistance. Furthermore, the two iso-enzymes are differentially inhibited by estrogens, polyamines and neuroleptics, and also differ in heat-lability. To elucidate the molecular mechanisms that underlie these different regulation patterns of the two iso-enzymes (and consequently the evolutionary adaptation of hGDH2 to a new functional role), we have performed mutagenesis at sites of difference in their amino acid sequence. Results showed that the low basal activity, heat-lability and estrogen sensitivity of hGDH2 could be, at least partially, ascribed to the Arg443Ser evolutionary change, whereas resistance to GTP inhibition has been attributed to the Gly456Ala change. Other amino acid substitutions studied thus far cannot explain all the remaining functional differences between the two iso-enzymes. Also, the Arg443Ser/Gly456Ala double mutation in hGDH1 approached the properties of wild-type hGDH2, without being identical to it. The insights into the structural mechanism of enzymatic regulation and the implications in cell biology provided by these findings are discussed.

Multiple Forms of Glutamate Dehydrogenase in Animals: Structural Determinants and Physiological Implications

“…ThDP binding to the ADP site, competitive with that of ADP, is consistent with certain structural similarities between the thiamine compounds and nucleotides, in particular, adenosine (Figure 4), and binding of thiamine to some adenosine sites [117]. In this regard, different regulation of the two GDH isoenzymes with nucleotides [50,101,110], discussed in Section 5, favors their different reactivity also to the thiamine compounds.…”

“…Other amino acid substitutions studied thus far cannot explain all the functional differences between the two isoenzymes [110]. For instance, the Arg443Ser/Gly456Ala double mutation in hGDH1 did not result in the properties of wild-type hGDH2 [50].…”

Amino Acid Catabolism in Alzheimer’s Disease Brain: Friend or Foe?

“…Glutamine synthetase activity has been shown to decline in AD brain likely through oxidative inactivation [ 96 ], although protein levels have been shown to increase in the prefrontal cortex and CSF. Mitochondrial GLUD1 is negatively regulated by the GTP formed from citric acid cycle function, while GLUD2 is relatively unaffected by guanine nucleotides but is positively regulated by ADP and branched chain amino acids [ 97 ]. The negative regulation of GLUD1 when energy levels are high allows for preservation of glutamate levels needed for neurotransmission as well as preventing the toxic buildup of ammonia.…”

Glutamate production from ammonia via glutamate dehydrogenase 2 activity supports cancer cell proliferation under glutamine depletion

“…From these results, we hypothesized that MCF7 cells could proliferate by utilizing ammonia produced from glutamine when the available glutamine level decreased. To understand whether ammonia plays a critical role in cell proliferation, presumably as a nitrogen donor, we incubated MCF7 cells with or without ammonium sulfate under glutamine-depleted 8 conditions. The addition of ammonium sulfate partially restored MCF7 cell proliferation in the absence of glutamine (Fig.…”

“…GDH normally catalyzes glutamate to α-KG and ammonia, employing nicotinamide adenine dinucleotide phosphate (NADP + ) and nicotinamide adenine dinucleotide (NAD + ) as cofactors. However, GDH can also catalyze reductive amination to produce glutamate from α-KG and ammonia, employing NADPH and NADH as cofactors, depending on the environment [8,9]. Humans possess two GDH isoforms -GDH1 and GDH2 (encoded by the GLUD1 and GLUD2 genes, respectively)- 4 that have high sequence similarity.…”