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

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.

Side-chain interactions in the regulatory domain of human glutamate dehydrogenase determine basal activity and regulation.

Glutamate Dehydrogenase (GDH) is central to the metabolism of glutamate, a major excitatory transmitter in mammalian central nervous system (CNS). hGDH1 is activated by ADP and L-leucine and powerfully inhibited by GTP. Besides this housekeeping hGDH1, duplication led to an hGDH2 isoform that is expressed in the human brain dissociating its function from GTP control. The novel enzyme has reduced basal activity (4-6% of capacity) while remaining remarkably responsive to ADP/L-leucine activation. While the molecular basis of this evolutionary adaptation remains unclear, substitution of Ser for Arg443 in hGDH1 is shown to diminish basal activity (< 2% of capacity) and abrogate L-leucine activation. To explore whether the Arg443Ser mutation disrupts hydrogen bonding between Arg443 and Ser409 of adjacent monomers in the regulatory domain ('antenna'), we replaced Ser409 by Arg or Asp in hGDH1. The Ser409Arg-1 change essentially replicated the Arg443Ser-1 mutation effects. Molecular dynamics simulation predicted that Ser409 and Arg443 of neighboring monomers come in close proximity in the open conformation and that introduction of Ser443-1 or Arg409-1 causes them to separate with the swap mutation (Arg409/Ser443) reinstating their proximity. A swapped Ser409Arg/Arg443Ser-1 mutant protein, obtained in recombinant form, regained most of the wild-type hGDH1 properties. Also, when Ser443 was replaced by Arg443 in hGDH2 (as occurs in hGDH1), the Ser443Arg-2 mutant acquired most of the hGDH1 properties. Hence, side-chain interactions between 409 and 443 positions in the 'antenna' region of hGDHs are crucial for basal catalytic activity, allosteric regulation, and relative resistance to thermal inactivation.

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.

Linking pesticide exposure and dementia: what is the evidence?

There has been a steep increase in the prevalence of dementia in recent decades, which has roughly followed an increase in pesticide use some decades earlier, a time when it is probable that current dementia patients could have been exposed to pesticides. This raises the question whether pesticides contribute to dementia pathogenesis. Indeed, many studies have found increased prevalence of cognitive, behavioral and psychomotor dysfunction in individuals chronically exposed to pesticides. Furthermore, evidence from recent studies shows a possible association between chronic pesticide exposure and an increased prevalence of dementia, including Alzheimer's disease (AD) dementia. At the cellular and molecular level, the mechanism of action of many classes of pesticides suggests that these compounds could be, at least partly, accountable for the neurodegeneration accompanying AD and other dementias. For example, organophosphates, which inhibit acetylcholinesterase as do the drugs used in treating AD symptoms, have also been shown to lead to microtubule derangements and tau hyperphosphorylation, a hallmark of AD. This emerging association is of considerable public health importance, given the increasing dementia prevalence and pesticide use. Here we review the epidemiological links between dementia and pesticide exposure and discuss the possible pathophysiological mechanisms and clinical implications of this association.

Role of paraoxonase 1 (PON1) in organophosphate metabolism: implications in neurodegenerative diseases.

Organophosphate pesticides are a class of compounds that are widely used in agricultural and rural areas. Paraoxonase 1 (PON1) is a phase-I enzyme that is involved in the hydrolysis of organophosphate esters. Environmental poisoning by organophosphate compounds has been the main driving force of previous research on PON1 enzymes. Recent discoveries in animal models have revealed the important role of the enzyme in lipid metabolism. However although PON1 function is well established in experimental models, the contribution of PON1 in neurodegenerative diseases remains unclear. In this minireview we summarize the involvement of PON1 genotypes in the occurrence of Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. A brief overview of latest epidemiological studies, regarding the two most important PON1 coding region polymorphisms PON1-L55M and PON1-Q192R is presented. Positive and negative associations of PON1 with disease occurrence are reported. Notably the MM and RR alleles contribute a risk enhancing effect for the development of some neurodegenerative diseases, which may be explained by the reduced lipoprotein free radical scavenging activity that may give rise to neuronal damage, through distinct mechanism. Conflicting findings that fail to support this postulate may represent the human population ethnic heterogeneity, different sample size and environmental parameters affecting PON1 status. We conclude that further epidemiological studies are required in order to address the exact contribution of PON1 genome in combination with organophosphate exposure in populations with neurodegenerative diseases.

A case report of motor neuron disease in a patient showing significant level of DDTs, HCHs and organophosphate metabolites in hair as well as levels of hexane and toluene in blood.

Motor neuron disease is a devastating neurodegenerative condition, with the majority of sporadic, non-familial cases being of unknown etiology. Several epidemiological studies have suggested that occupational exposure to chemicals may be associated with disease pathogenesis. We report the case of a patient developing progressive motor neuron disease, who was chronically exposed to pesticides and organic solvents. The patient presented with leg spasticity and developed gradually clinical signs suggestive of amyotrophic lateral sclerosis, which was supported by the neurophysiologic and radiological findings. Our report is an evidence based case of combined exposure to organochlorine (DDTs), organophosphate pesticides (OPs) and organic solvents as confirmed by laboratory analysis in samples of blood and hair confirming systematic exposure. The concentration of non-specific dialkylphosphates metabolites (DAPs) of OPs in hair (dimethyphopshate (DMP) 1289.4 pg/mg and diethylphosphate (DEP) 709.4 pg/mg) and of DDTs (opDDE 484.0 pg/mg, ppDDE 526.6 pg/mg, opDDD 448.4 pg/mg, ppDDD+opDDT 259.9 pg/mg and ppDDT 573.7 pg/mg) were considerably significant. Toluene and n-hexane were also detected in blood on admission at hospital and quantified (1.23 and 0.87 µg/l, respectively), while 3 months after hospitalization blood testing was found negative for toluene and n-hexane and hair analysis was provided decrease levels of HCHs, DDTs and DAPs.

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.

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.

The human GLUD2 glutamate dehydrogenase: localization and functional aspects.

In all mammals, glutamate dehydrogenase (GDH), an enzyme central to the metabolism of glutamate, is encoded by a single gene (GLUD1 in humans) which is expressed widely (housekeeping). Humans and other primates also possess a second gene, GLUD2, which encodes a highly homologous GDH isoenzyme (hGDH2) expressed predominantly in retina, brain and testis. There is evidence that GLUD1 was retro-posed <23 million years ago to the X chromosome, where it gave rise to GLUD2 through random mutations and natural selection. These mutations provided the novel enzyme with unique properties thought to facilitate its function in the particular milieu of the nervous system. hGDH2, having been dissociated from GTP control (through the Gly456Ala change), is mainly regulated by rising levels of ADP/l-leucine. To achieve full-range regulation by these activators, hGDH2 needs to set its basal activity at low levels (<10% of full capacity), a property largely conferred by the evolutionary Arg443Ser change. Studies of structure/function relationships have identified residues in the regulatory domain of hGDH2 that modify basal catalytic activity and regulation. In addition, enzyme concentration and buffer ionic strength can influence basal enzyme activity. While mature hGDH1 and hGDH2 isoproteins are highly homologous, their predicted leader peptide sequences show a greater degree of divergence. Study of the subcellular sites targeted by hGDH2 in three different cultured cell lines using a GLUD2/EGFP construct revealed that hGDH2 localizes mainly to mitochondria and to a lesser extent to the endoplasmic reticulum of these cells. The implications of these findings for the potential role of this enzyme in the biology of the nervous system in health and disease are discussed.

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.

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.