The odyssey of a young gene: structure-function studies in human glutamate dehydrogenases reveal evolutionary-acquired complex allosteric regulation mechanisms.

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.

Estrogen modification of human glutamate dehydrogenases is linked to enzyme activation state.

Mammalian glutamate dehydrogenase (GDH) is a housekeeping enzyme central to the metabolism of glutamate. Its activity is potently inhibited by GTP (IC(50) = 0.1-0.3 µM) and thought to be controlled by the need of the cell in ATP. Estrogens are also known to inhibit mammalian GDH, but at relatively high concentrations. Because, in addition to this housekeeping human (h) GDH1, humans have acquired via a duplication event an hGDH2 isoform expressed in human cortical astrocytes, we tested here the interaction of estrogens with the two human isoenzymes. The results showed that, under base-line conditions, diethylstilbestrol potently inhibited hGDH2 (IC(50) = 0.08 ± 0.01 µM) and with ∼18-fold lower affinity hGDH1 (IC(50) = 1.67 ± 0.06 µM; p < 0.001). Similarly, 17ß-estradiol showed a ∼18-fold higher affinity for hGDH2 (IC(50) = 1.53 ± 0.24 µM) than for hGDH1 (IC(50) = 26.94 ± 1.07 µM; p < 0.001). Also, estriol and progesterone were more potent inhibitors of hGDH2 than hGDH1. Structure/function analyses revealed that the evolutionary R443S substitution, which confers low basal activity, was largely responsible for sensitivity of hGDH2 to estrogens. Inhibition of both human GDHs by estrogens was inversely related to their state of activation induced by ADP, with the slope of this correlation being steeper for hGDH2 than for hGDH1. Also, the study of hGDH1 and hGDH2 mutants displaying different states of activation revealed that the affinity of estrogen for these enzymes correlated inversely (R = 0.99; p = 0.0001) with basal catalytic activity. Because astrocytes are known to synthesize estrogens, these hormones, by interacting potently with hGDH2 in its closed state, may contribute to regulation of glutamate metabolism in brain.

Mutations in human GLUD2 glutamate dehydrogenase affecting basal activity and regulation.

Glutamate dehydrogenase (GDH) in human exists in GLUD1 and GLUD2 gene-encoded isoforms (hGDH1 and hGDH2, respectively), differing in their regulation and tissue expression pattern. Whereas hGDH1 is subject to GTP control, hGDH2 uses for its regulation, a novel molecular mechanism not requiring GTP. This is based on the ability of hGDH2 to maintain a baseline activity of <10% of its capacity subject to full activation by rising ADP/L-leucine levels. Here we studied further the molecular mechanisms regulating hGDH2 function by creating and analyzing hGDH2 mutants harboring single amino acid substitutions in the regulatory domain (antenna, pivot helix) of the protein. Five hGDH2 mutants were obtained: two with an amino acid change (Gln441Arg, Ser445Leu) in the antenna, two (Lys450Glu, His454Tyr) in the pivot helix, and one (Ser448Pro) in the junction between the two structures. Functional analyses revealed that, while the antenna mutations increased basal enzyme activity without affecting its allosteric properties, the pivot helix mutations drastically reduced basal activity and impaired enzyme regulation. On the other hand, the Ser448Pro mutation reduced basal activity but did not alter allosteric regulation. Also, compared with wild-type hGDH2, the antenna mutants were relatively thermostable, whereas the pivot helix mutants were extremely heat labile. Hence, the present data further our understanding of the molecular mechanisms involved in the function and stability of hGDH2, an enzyme thought to be of importance for nerve tissue biology.

Gain-of-function variant in GLUD2 glutamate dehydrogenase modifies Parkinson's disease onset.

Parkinson's disease (PD), a common neurodegenerative disorder characterized by progressive loss of dopaminergic neurons and their terminations in the basal ganglia, is thought to be related to genetic and environmental factors. Although the pathophysiology of PD neurodegeneration remains unclear, protein misfolding, mitochondrial abnormalities, glutamate dysfunction and/or oxidative stress have been implicated. In this study, we report that a rare T1492G variant in GLUD2, an X-linked gene encoding a glutamate dehydrogenase (a mitochondrial enzyme central to glutamate metabolism) that is expressed in brain (hGDH2), interacted significantly with age at PD onset in Caucasian populations. Individuals hemizygous for this GLUD2 coding change that results in substitution of Ala for Ser445 in the regulatory domain of hGDH2 developed PD 6-13 years earlier than did subjects with other genotypes in two independent Greek PD groups and one North American PD cohort. However, this effect was not present in female PD patients who were heterozygous for the DNA change. The variant enzyme, obtained by substitution of Ala for Ser445, showed an enhanced basal activity that was resistant to GTP inhibition but markedly sensitive to modification by estrogens. Thus, a gain-of-function rare polymorphism in hGDH2 hastens the onset of PD in hemizygous subjects, probably by damaging nigral cells through enhanced glutamate oxidative dehydrogenation. The lack of effect in female heterozygous PD patients could be related to a modification of the overactive variant enzyme by estrogens.

Properties and molecular evolution of human GLUD2 (neural and testicular tissue-specific) glutamate dehydrogenase.

Glutamate dehydrogenase (GDH) is an enzyme central to the metabolism of glutamate that also plays a role in cellular energetics. In the human, GDH exists in a housekeeping isoenzyme (hGDH1) encoded by the GLUD1 gene and a neural and testicular tissue-specific isoform (hGDH2) encoded by the GLUD2 gene. There is evolutionary evidence that the GLUD1 was retroposed to the X chromosome in the ape ancestor (>23 million years ago), where it gave rise to GLUD2 through random mutations and directional selection. In the human, the two mature GDH isoproteins are highly homologous, differing in only 16 of their 505 amino acid residues. Functional analyses of highly purified recombinant wild-type hGDH2 revealed that this adaptive evolution dissociated the enzyme from GTP control, permitted regulation almost entirely by ADP and/or L-leucine, and fine-tuned its activity to the relatively low cellular pH that occurs in synaptic astrocytes during excitatory transmission. Study of structure-function relationships, using site-directed mutagenesis of GLUD1 at single sites differing from GLUD2, showed that the Arg443Ser and the Gly456Ala change reproduced some, but not all, of the properties of hGDH2. In addition, we created a double hGDH1 mutant that had both Arg443Ser and Gly456Ala in the same polypeptide chain. Functional analyses revealed that the doubly mutated enzyme did not acquire all the characteristics of the wild-type hGDH2. Hence, additional amino acid changes, acting in concert with Arg443Ser and Gly456Ala, ought to be responsible the unique properties of the brain-specific human isoenzyme.

Properties and molecular evolution of human GLUD2 (neural and testicular tissue-specific) glutamate dehydrogenase.

Glutamate dehydrogenase (GDH) is an enzyme central to the metabolism of glutamate that also plays a role in cellular energetics. In the human, GDH exists in a housekeeping isoenzyme (hGDH1) encoded by the GLUD1 gene and a neural and testicular tissue-specific isoform (hGDH2) encoded by the GLUD2 gene. There is evolutionary evidence that the GLUD1 was retroposed to the X chromosome in the ape ancestor (<23 million years ago), where it gave rise to GLUD2 through random mutations and directional selection. In the human, the two mature GDH isoproteins are highly homologous, differing in only 16 of their 505 amino acid residues. Functional analyses of highly purified recombinant wild-type hGDH2 revealed that this adaptive evolution dissociated the enzyme from GTP control, permitted regulation almost entirely by ADP and/or L-leucine, and fine-tuned its activity to the relatively low cellular pH that occurs in synaptic astrocytes during excitatory transmission. Study of structure-function relationships, using site-directed mutagenesis of GLUD1 at single sites differing from GLUD2, showed that the Arg443Ser and the Gly456Ala change reproduced some, but not all, of the properties of hGDH2. In addition, we created a double hGDH1 mutant that had both Arg443Ser and Gly456Ala in the same polypeptide chain. Functional analyses revealed that the doubly mutated enzyme did not acquire all the characteristics of the wild-type hGDH2. Hence, additional amino acid changes, acting in concert with Arg443Ser and Gly456Ala, ought to be responsible the unique properties of the brain-specific human isoenzyme.