**1. Introduction**

288 Dehydrogenases

(January 1997), pp. 21-27, ISSN 0022-4251

2001), pp. 415-420, ISSN 0916-8818

2007), pp. 429–433, ISSN 0021-9541

Issue 2 (November 2011), pp. 137– 142, ISSN 0378-4274

No. 6, (June 2010), pp. 1019–1020, ISSN 1470-1626

ISSN 1471-2970

ISSN 0890-6238

Muller, J.; Rajpert-De Meyts, E.; Scheike, T.; Sharpe, R.; Sumpter, J. & Skakkebaek, N.E. (1996) Male reproductive health and environmental xenoestrogens. *Environmental Health Perspectives*, Vol. 104, Suppl. 4, (August 1996), pp. 741–803, ISSN 0091-6765 Tsubota, T.; Howell-Skalla, L.; Nitta, H.; Osawa, Y.; Mason, J.I.; Meiers, P.G.; Nelson, R.A. & Bahr, J.M. (1997) Seasonal changes in spermatogenesis and testicular steroidogenesis in the male black bear *Ursus americanus*. *Journal of Reproduction and Fertility*, Vol. 109, No. 1,

Tsubota, T.; Nagashima, T.; Kohyama, K.; Maejima, K.; Murase, T. & Kita, I. (2001) Seasonal changes in testicular steroidogenesis and spermatogenesis in a northern fur seal, *Callorhinus ursinus*. *Journal of Reproduction and Development*, Vol. 47, No. 6, (December

Ulloa-Aguirre, A.; Bassol, S.; Poo, J.; Mendez, J.P.; Mutchinick, O.; Robles, C. & Perez-Palacios, G. (1985) Endocrine and biochemical studies in a 46,XY phenotypically male infant with 17-ketosteroid reductase deficiency. *Journal of Clinical Endocrinology &* 

Vaithinathan, S.; Saradha, B. & Mathur, P.P. (2008) Transient inhibitory effect of methoxychlor on testicular steroidogenesis in rat: an *in vivo* study. *Archives in* 

Verhoeven, G.; Willems, A.; Denolet, E.; Swinnen, J.V, & De Gendt, K. (2010) Androgens and spermatogenesis: lessons from transgenic mouse models. *Philosophical Transactions of the Royal Society Series B, Biological Sciences,* Vol. 27, No. 1546, (May 2010), pp. 1537-1556,

Victor-Costa, A.B.; Carozzi Bandeira, S.M.; Oliveira, A.G.; Bohórquez Mahecha, G.A. & Oliveira, C.A. (2010) Changes in testicular morphology and steroidogenesis in adult rats exposed to atrazine. *Reproductive Toxicology*, Vol. 29, No. 3, (June 2010), pp. 323–331,

Wang, R-S.; Yeh, S.; Tzeng, C-R. & Chang, C. (2009) Androgen receptor roles in spermatogenesis and fertility: lessons from testicular cell-specific androgen receptor knockout mice. *Endocrine Reviews*, Vol. 30, No. 2, (April 2009), pp. 119-132, ISSN 0163-769X Wisner, J.R Jr. & Gomes, W.R. (1978) Influence of experimental cryptorchidism on cholesterol side-chain cleavage enzyme and delta5-3beta-hydroxysteroid dehydrogenase activities

in rat testes. *Steroids*, Vol. 31, No. 2, (February 1978), pp. 189–203, ISSN 0585-2617 Wu, X.; Wan, S. & Lee M.M. (2007) Key factors in the regulation of fetal and postnatal Leydig cell development. *Journal of Cellular Physiology*, Vol. 213, No. 2, (November

Ye, L.; Zhao, B.; Hu, G.; Chu, Y. & Ge, R-S. (2011) Inhibition of human and rat testicular steroidogenic enzyme activities by bisphenol A. *Toxicology Letters*, Vol. 30, No. 207,

Zirkin, B.R. (2010) Where do adult Leydig cells come from? *Biology of Reproduction*, Vol. 82,

Zirkin, B.R. & Chen, H. (2000) Regulation of Leydig cell steroidogenic function during aging. *Biology of Reproduction*, Vol. 63, No. 4, (October 2000), pp. 977–981, ISSN 0006-3363

*Metabolism*, Vol. 60, No. 4, (April 1985), pp. 639–643, ISSN 1521-690X

*Toxicology*, Vol. 82, No. 11, (April 2008), pp. 833–839, ISSN 0340-5761

Glutamate dehydrogenase (GDH) is present in all domains of life and is one of the most extensively studied enzymes at the biochemical and structural levels. These enzymes are generally reversible and catalyse either the reductive amination of 2-oxoglutarate (2-OG) to yield glutamate using NAD(P) as a cofactor, or the oxidative deamination of glutamate [1] (Fig. 1). Because of the reaction it catalyses, the main role of GDH is glutamate catabolism and ammonium assimilation. However, other physiological roles for GDH have been described in some organisms, as we will see below.

**Figure 1.** Reaction catalysed by glutamate dehydrogenase

The synthesis of both glutamate and glutamine are key steps in the cell metabolism in all organisms, because they represent the only means of incorporating inorganic nitrogen into carbon backbones. Inorganic nitrogen is assimilated in the form of ammonium, which is incorporated as an amino group to glutamate or an amido group to glutamine. These amino acids in turn act as amino group donors for the synthesis of most nitrogen-containing compounds in the cell. In particular, the amino group of glutamate is used in the synthesis

> © 2012 Santero et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of purines, pyrimidines, amino sugars, histidine, tryptophan, asparagine, NAD and *p*aminobenzoate. Therefore glutamate is a key element in the nitrogen flow, as it plays a role of nitrogen donor and acceptor.

Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance 291

that yield amino groups into the urea cycle are localized in the mitochondria. In the liver, glutamate is the source of excess ammonium release, and the concentration of glutamate modulates the rate of ammonia detoxification into urea. In pancreatic β-cells, the GDH is involved in insulin homeostasis, and oxidation of glutamate mediates amino acid-stimulated insulin secretion [8]. In the central nervous system, glutamate serves as a neurotransmitter and also as the precursor of the inhibitory neurotransmitter γ-aminobutyric acid (GABA), as well as glutamine, a potential mediator of hyperammonemic neurotoxicity [7]. Also, excessive glutamate signalling can lead to excitotoxicity, a phenomenon where over-activation of glutamate receptors initiates neuronal death [9]. The clinical importance of glutamate metabolism in β-pancreatic cells has been highlighted by the recent discovery of a dominantly expressed defect in glutamate metabolism, the hyperinsulinism/hypermmonemia syndrome (HHS). HHS was one of the first diseases that clearly linked GDH regulation to insulin and ammonia homeostasis [10]. Affected children suffer from recurrent hypoglycemia due to inappropriate secretion of insulin [10-12]. This syndrome is caused by the loss of the human

**Figure 2.** Flow of nitrogen in the biosphere. Molecular nitrogen, nitrites and nitrates are reduced to ammonium and assimilated by microorganisms and plants, whilst higher eukaryotes assimilate these

Several GDH classifications have been done according to their size, oligomerisation state, coenzyme specificity or organism, among others. According to their cofactor specificity, there are three basic types of GDH: those that are cofactor specific for NAD (EC 1.4.1.2), those that are specific for NADP (EC 1.4.1.4) and those that can use either cofactor (EC 1.4.1.3) (dual coenzyme-specific GDHs). Lower eukaryotes and prokaryotes usually have GDHs that only function with one coenzyme whilst the enzymes that have dual coenzyme

glutamate dehydrogenase allosteric regulation (see below).

nitrogenated compounds as protein in their diets.

**2. Classification, evolution and structure of GDHs** 

Glutamate can be synthesized by two alternative routes: one involves catalysis of GDH in the aminating direction, but ammonium assimilation is also possible by the participation of two enzymes: glutamine synthetase (GS), and glutamate synthase, also named glutamine oxoglutarate aminotransferase or GOGAT (Figure 2). The disadvantage of this pathway is its extra energy requirement. Although GDH catalyses the reductive amination of 2-OG, it is noteworthy that because of its overall high Km for ammonium, this reaction can only be used for the synthesis of glutamate when the ammonium concentration is high (>1 mM). When the ammonium concentration is lower, ammonia is incorporated to glutamate mainly via the GS-GOGAT pathway. Generally, GDH activity is not necessary for cell growth, since most organisms can synthesize glutamate from glutamine and 2-OG using GOGAT. In fact, some bacteria naturally lack GDH and are neither glutamate auxotrophs nor affected in nitrogen assimilation. While the amination reaction provides nitrogen required for many biosynthetic pathways, the oxidative deamination reaction of GDH provides carbon to the tricarboxylic acid cycle (TCA) by conversion of L-glutamate to 2-OG and probably contributes to balancing the glutamine to glutamate ratio.

Plants and microorganisms can utilise several inorganic nitrogen sources with different oxidation states such as N2 (by nitrogen-fixing bacteria and archaea), nitrate or nitrite, by reducing them to ammonium, which is subsequently assimilated. After formation of glutamate, the α-amino group can be transferred to a wide variety of 2-oxo acceptors to give rise to amino acids. Also, the α-amino group can be transferred again to glutamate, when 2 oxoglutarate and other amino acids are available. These reactions are carried out by the reversible activity of aminotransferases (EC 2.6.1.x) (Figure 2). Plants and microorganisms can synthesize all carbon skeletons for their amino acids and incorporate the amino group to them by transamination using glutamine and glutamate as nitrogen donors. Incorporation of ammonium in animals also occurs through the GDH and GS/GOGAT pathways. However, higher organisms are unable to reduce oxidized forms of nitrogen to ammonium, to synthesize the structures of some branched or aromatic amino acids such as tryptophan or phenylalanine, or to incorporate sulphur into covalently bonded structures. They are, therefore, totally dependent on other organisms to convert oxidized forms of nitrogen into forms available for the organism, as well as to provide some essential amino acids (Figure 2). These are supplied in the diet or are provided by bacteria from the intestinal tract.

In plants and microorganisms, the physiological roles of GDH include nitrogen assimilation [2], glutamate catabolism [1, 3], but also osmotic balance [4] and tolerance to high temperatures [5, 6]. In vertebrates, multiple biochemical pathways involve glutamate, which is also used as a neurotransmitter. The imbalance of the GDH activity may lead to disturbances of clinical relevance for humans [7]. Free ammonia is highly toxic to organisms that excrete urea as the main nitrogenous waste such as mammals, fish and adult amphibians, leading to inhibition of brain respiration and an excess ketone body formation from acetyl-CoA in the liver. To prevent these deleterious effects, GDH and some of the other enzymes that yield amino groups into the urea cycle are localized in the mitochondria. In the liver, glutamate is the source of excess ammonium release, and the concentration of glutamate modulates the rate of ammonia detoxification into urea. In pancreatic β-cells, the GDH is involved in insulin homeostasis, and oxidation of glutamate mediates amino acid-stimulated insulin secretion [8]. In the central nervous system, glutamate serves as a neurotransmitter and also as the precursor of the inhibitory neurotransmitter γ-aminobutyric acid (GABA), as well as glutamine, a potential mediator of hyperammonemic neurotoxicity [7]. Also, excessive glutamate signalling can lead to excitotoxicity, a phenomenon where over-activation of glutamate receptors initiates neuronal death [9]. The clinical importance of glutamate metabolism in β-pancreatic cells has been highlighted by the recent discovery of a dominantly expressed defect in glutamate metabolism, the hyperinsulinism/hypermmonemia syndrome (HHS). HHS was one of the first diseases that clearly linked GDH regulation to insulin and ammonia homeostasis [10]. Affected children suffer from recurrent hypoglycemia due to inappropriate secretion of insulin [10-12]. This syndrome is caused by the loss of the human glutamate dehydrogenase allosteric regulation (see below).

290 Dehydrogenases

of nitrogen donor and acceptor.

balancing the glutamine to glutamate ratio.

of purines, pyrimidines, amino sugars, histidine, tryptophan, asparagine, NAD and *p*aminobenzoate. Therefore glutamate is a key element in the nitrogen flow, as it plays a role

Glutamate can be synthesized by two alternative routes: one involves catalysis of GDH in the aminating direction, but ammonium assimilation is also possible by the participation of two enzymes: glutamine synthetase (GS), and glutamate synthase, also named glutamine oxoglutarate aminotransferase or GOGAT (Figure 2). The disadvantage of this pathway is its extra energy requirement. Although GDH catalyses the reductive amination of 2-OG, it is noteworthy that because of its overall high Km for ammonium, this reaction can only be used for the synthesis of glutamate when the ammonium concentration is high (>1 mM). When the ammonium concentration is lower, ammonia is incorporated to glutamate mainly via the GS-GOGAT pathway. Generally, GDH activity is not necessary for cell growth, since most organisms can synthesize glutamate from glutamine and 2-OG using GOGAT. In fact, some bacteria naturally lack GDH and are neither glutamate auxotrophs nor affected in nitrogen assimilation. While the amination reaction provides nitrogen required for many biosynthetic pathways, the oxidative deamination reaction of GDH provides carbon to the tricarboxylic acid cycle (TCA) by conversion of L-glutamate to 2-OG and probably contributes to

Plants and microorganisms can utilise several inorganic nitrogen sources with different oxidation states such as N2 (by nitrogen-fixing bacteria and archaea), nitrate or nitrite, by reducing them to ammonium, which is subsequently assimilated. After formation of glutamate, the α-amino group can be transferred to a wide variety of 2-oxo acceptors to give rise to amino acids. Also, the α-amino group can be transferred again to glutamate, when 2 oxoglutarate and other amino acids are available. These reactions are carried out by the reversible activity of aminotransferases (EC 2.6.1.x) (Figure 2). Plants and microorganisms can synthesize all carbon skeletons for their amino acids and incorporate the amino group to them by transamination using glutamine and glutamate as nitrogen donors. Incorporation of ammonium in animals also occurs through the GDH and GS/GOGAT pathways. However, higher organisms are unable to reduce oxidized forms of nitrogen to ammonium, to synthesize the structures of some branched or aromatic amino acids such as tryptophan or phenylalanine, or to incorporate sulphur into covalently bonded structures. They are, therefore, totally dependent on other organisms to convert oxidized forms of nitrogen into forms available for the organism, as well as to provide some essential amino acids (Figure

2). These are supplied in the diet or are provided by bacteria from the intestinal tract.

In plants and microorganisms, the physiological roles of GDH include nitrogen assimilation [2], glutamate catabolism [1, 3], but also osmotic balance [4] and tolerance to high temperatures [5, 6]. In vertebrates, multiple biochemical pathways involve glutamate, which is also used as a neurotransmitter. The imbalance of the GDH activity may lead to disturbances of clinical relevance for humans [7]. Free ammonia is highly toxic to organisms that excrete urea as the main nitrogenous waste such as mammals, fish and adult amphibians, leading to inhibition of brain respiration and an excess ketone body formation from acetyl-CoA in the liver. To prevent these deleterious effects, GDH and some of the other enzymes

**Figure 2.** Flow of nitrogen in the biosphere. Molecular nitrogen, nitrites and nitrates are reduced to ammonium and assimilated by microorganisms and plants, whilst higher eukaryotes assimilate these nitrogenated compounds as protein in their diets.

### **2. Classification, evolution and structure of GDHs**

Several GDH classifications have been done according to their size, oligomerisation state, coenzyme specificity or organism, among others. According to their cofactor specificity, there are three basic types of GDH: those that are cofactor specific for NAD (EC 1.4.1.2), those that are specific for NADP (EC 1.4.1.4) and those that can use either cofactor (EC 1.4.1.3) (dual coenzyme-specific GDHs). Lower eukaryotes and prokaryotes usually have GDHs that only function with one coenzyme whilst the enzymes that have dual coenzyme

specificity are commonly found in higher eukaryotes. However, some dual-GDHs, have also been described in prokaryotes [5, 13-15]. Glutamate dehydrogenases from non-vertebrate animals differ from the GDHs of vertebrates in that they are mono-coenzyme specific and are not regulated by nucleotides [16]. In higher plants GDH is ubiquitous and also very abundant. A number of isozymes are usually present in a single species, some of them being inducible, which correlate with their abundance depending on environmental or nutritional conditions [17]. GDHs have also been characterized from a number of eukaryotic microorganisms with different coenzyme specificity such as fungi, (NAD+ or NADP+) [18], algae (NAD+, NADP+ or dual), protozoa (NAD+ or NADP+) and also different intracellular localizations (i.e. cytoplasmic, mitochondrial or in the chloroplasts) [19].

Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance 293

*K***m NH4 (mM)** 

*K***m 2-OG (mM)** 

*K***m glu (mM) Ref.** 

**subunit number**

**Organism cofactor** 

*Halobacterium* 

*Thermococcus* 

**Eubacteria Gram negative**  *Capnocytophaga* 

*Janthinobacterium* 

*Psychrobacter sp* 

*Psychrobacter sp* 

**Gram positive** 

*Clostridium* 

*Corynebacterium* 

*Mycobacterium* 

*Mycobacterium* 

*Lactobacillus* 

*Thermococcus strain* 

**MW enzyme (KDa)** 

**MW subunit (KDa)** 

*halobium* NADP [33]

*AN1* NADP 204 47 4 15.5 1.7 9.12 [19] *Pyrococcus furiosus* NAD/NADP 270-290 48 6 6, 27b 0.33 0.6 [34]

*profundus* NADP 263 43 6 1.6, 22b 0.2, 0.87b 6.8 [6]

*ochraea* NAD 3.33 1.44 2.44 [35] *Escherichia coli B/r* NADP 300 50 6 1.1 0.64 [36] *Escherichia coli PA340* NADP 2.5 0.2 2.3 [37]

*lividum* NAD 1065 170 6 7.1 [24] *P. aeruginosa* NAD 180 4 15 1.6 [25, 38] *P. aeruginosa* NADP 110 7 1 [38, 39]

*TAD1* NAD 290 160 2 24.6 2.36 28.6 [27]

*TAD1* NADP 290 47 6 4 ND 67.4 [37] *Salmonella enterica* NADP 0.29 4 50 [40] *Thermus termophilus* NAD 289 46.5, 48a 6 [29, 41] *Thiobacillus novellus* NADP 130 50-55 2? 7.5 7.4 35.5 [42] *Thiobacillus novellus* NAD 120 50-55 2? 7.4, 0.5d 6.7, 0.67d 11.8, 13.3d [43]

*Bacillus macerans* NADP 2.2 0.38 [44] *Bacillus polymyxa* NADP 2.9 1.4 [45] *Bacillus subtilis* NAD 6 [46, 47]

*symbiosum* NAD 282 49 6 [48, 49]

*glutamicum* NADP 49 6? [50]

*smegmatis* NAD 180 4? [26, 51]

*smegmatis* NADP 245.5 40 6 33 5 62.5 [52]

*fermentum* NADP 300 50 6 6.76 5.6 79 [53]

According to the molecular weight of the monomer, three groups of GDHs can be distinguished: GDH50s (MW around 50 KDa), GDH115s (MW around 115 KDa) and GDH180s (MW around 180 KDa). All NADP and dual-GDHs reported so far belong to the GDH50 group, whereas there are representatives of NAD-GDHs in all of these groups. GDH115s have been found only in lower eukaryotes [20-22], whilst the largest GDHs are present only in bacteria. GDH180s were first identified in actinomycetes [23], but recently they have been also described in other Gram positive and Gram negative bacteria (see table 1). Most GDHs reported so far are homo-oligomeric enzymes, but they differ in the number of monomers that compose them. The majority of GDHs have a hexameric structure, as is the case of vertebrate GDHs, but tetrameric and even dimeric enzymes have also been found (see table 1 and references therein). Particularly, the most recently discovered family of prokaryotic GDH180s, have representatives of either hexameric [23, 24], tetrameric [25, 26] and dimeric [27] enzymes. In addition, a couple of GDH50s composed of two different subunits in the form of a hetero-hexamer have been reported [28, 29].

Analysis of the distribution pattern of *gdh* genes from all available sequenced genomes in the three domains of life reveal that all classes of *gdh* have been found in eubacteria and archaea and all but the large GDH have been found in eukaryotes. Both NAD+- and NADP+ dependent forms of GDH have been reported in higher plants, located in mitochondria and chloroplasts, respectively. The GDH enzyme is abundant in several plant organs, and its isoenzymatic profile can be influenced by dark stress, natural senescence or fruit ripening [30]. Genes coding for GDH seem to be absent in some archaeal genomes as well as in some of the smaller eubacterial and eukaryotic genomes. Among the organisms that do encode GDH, several genes coding for GDH may be found in the same genome. However, just one or two classes are represented, no genome has yet been shown to encode all classes.



Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance 293

292 Dehydrogenases

specificity are commonly found in higher eukaryotes. However, some dual-GDHs, have also been described in prokaryotes [5, 13-15]. Glutamate dehydrogenases from non-vertebrate animals differ from the GDHs of vertebrates in that they are mono-coenzyme specific and are not regulated by nucleotides [16]. In higher plants GDH is ubiquitous and also very abundant. A number of isozymes are usually present in a single species, some of them being inducible, which correlate with their abundance depending on environmental or nutritional conditions [17]. GDHs have also been characterized from a number of eukaryotic microorganisms with different coenzyme specificity such as fungi, (NAD+ or NADP+) [18], algae (NAD+, NADP+ or dual), protozoa (NAD+ or NADP+) and also different intracellular

According to the molecular weight of the monomer, three groups of GDHs can be distinguished: GDH50s (MW around 50 KDa), GDH115s (MW around 115 KDa) and GDH180s (MW around 180 KDa). All NADP and dual-GDHs reported so far belong to the GDH50 group, whereas there are representatives of NAD-GDHs in all of these groups. GDH115s have been found only in lower eukaryotes [20-22], whilst the largest GDHs are present only in bacteria. GDH180s were first identified in actinomycetes [23], but recently they have been also described in other Gram positive and Gram negative bacteria (see table 1). Most GDHs reported so far are homo-oligomeric enzymes, but they differ in the number of monomers that compose them. The majority of GDHs have a hexameric structure, as is the case of vertebrate GDHs, but tetrameric and even dimeric enzymes have also been found (see table 1 and references therein). Particularly, the most recently discovered family of prokaryotic GDH180s, have representatives of either hexameric [23, 24], tetrameric [25, 26] and dimeric [27] enzymes. In addition, a couple of GDH50s composed of two different

Analysis of the distribution pattern of *gdh* genes from all available sequenced genomes in the three domains of life reveal that all classes of *gdh* have been found in eubacteria and archaea and all but the large GDH have been found in eukaryotes. Both NAD+- and NADP+ dependent forms of GDH have been reported in higher plants, located in mitochondria and chloroplasts, respectively. The GDH enzyme is abundant in several plant organs, and its isoenzymatic profile can be influenced by dark stress, natural senescence or fruit ripening [30]. Genes coding for GDH seem to be absent in some archaeal genomes as well as in some of the smaller eubacterial and eukaryotic genomes. Among the organisms that do encode GDH, several genes coding for GDH may be found in the same genome. However, just one

or two classes are represented, no genome has yet been shown to encode all classes.

**MW subunit (KDa)** 

*Archaeglobus fulgidus* NADP 263 47 6 4 0.5 3.9 [31]

*halobium* NAD 450 20.2 4 [32]

**subunit number**

*K***m NH4 (mM)** 

*K***m 2-OG (mM)** 

*K***m glu (mM) Ref.** 

**MW enzyme (KDa)** 

localizations (i.e. cytoplasmic, mitochondrial or in the chloroplasts) [19].

subunits in the form of a hetero-hexamer have been reported [28, 29].

**Organism cofactor** 

**Archaea** 

*Halobacterium* 


Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance 295

of enzyme, by high ionic strength and also by allosteric ligands or cofactors [64]. Since the local concentration of GDH in some tissues is very high, aggregation might be a regulatory

Eukaryotic and prokaryotic GDHs share relatively high conservation in their primary and secondary structures [61] and the crystal structures of the bacterial [59, 65, 66] and mammalian forms [61, 63] of GDH confirm that the general architecture and the locations of the catalytically important residues have remained unchanged throughout evolution. Each subunit in this multimeric enzyme is organised into two domains separated by a deep cleft. One domain directs the self-assembly of the molecule into a hexameric oligomer with 32 symmetry. The other domain is structurally similar to the classical pyridine nucleotidebinding domain but with the direction of one of the β-strands reversed. Upon glutamate binding, the enzyme can adopt different conformations by flexing about the cleft between its two domains. NAD+ binds in an extended conformation with the nicotinamide moiety buried deep in the cleft between the two domains [59, 61, 63, 65, 66]. The bottom domains of each trimer make wide contacts with each other, while the NAD+-binding domains bearing

The largest structural difference between mammalian and bacterial GDH is the *antenna*, which has a helix-loop-helix conformation. The *antenna* ascends from the NAD+-binding domain surface via a long, 23-residue helix and then descends back with a random coil structure. The helices of the "*antenna*" domains in each subunit of the trimer wrap around each other with a right-handed twist to form the core of the antenna protrusion. Extensive contacts between "antennae" may represent hexamer interactions in solution and, perhaps, with other enzymes within the mitochondrial matrix [61]. The fact that *antennae* are only found in the forms of GDH that are allosterically regulated by numerous ligands leads to the interpretation that it plays a major part in this regulation. In contrast to the extensive allosteric homotropic and heterotropic regulation observed in mammalian GDH (see below),

As a reversible enzyme, GDH has the potential for catalysing the reaction in the biosynthetic, aminating direction, or in the catabolic, deaminating direction. The actual physiological reaction of each GDH depends on several factors, as the kinetic constants of the enzyme for its different substrates or the environment where the cell is developed may widely vary. In general, NADP+-GDHs usually operate in the biosynthetic direction, that is, synthesizing glutamate by the assimilation of ammonia into 2-OG [6, 31, 39, 40, 45, 67, 68], whereas NAD+-GDHs have primarily a catabolic function, yielding ammonia and 2-oxoglutarate from the oxidative catabolism of glutamate [23, 24, 39, 68] (Table 1). Sometimes, both enzymes are present in the same organism, and play a different physiological role due to their different kinetic properties or their different time or place of expression [27, 51, 69, 70]. *Pseudomonas aeruginosa* and presumably other members of the genus *Pseudomonas* have a NADP+-specific and a NAD+-specific GDH, and it has been

the nucleotide-binding motif are poised at the top of the structure.

bacterial forms of GDH are relatively unregulated.

**3. GDH enzymology and physiological role** 

mechanism of the activity *in vivo*.

a NAD+-GDH of *Thermus thermophilus* is a heterohexamer composed by two types of subunits: GdhA (46, 5 KDa) and GdhB (48 KDa)

b The Km depends on the substrate concentration

c The kinetic constants determined for each cofactor in enzymes with dual cofactor specificity are separated by slashes

d The kinetic constants of NAD+-GDH of *T. novellus* are different depending on the presence of AMP

**Table 1.** Some characteristics of selected prokaryotic GDHs

The distribution of *gdh* genes does not show any strong pattern that correlate with the phylogeny [56]. It was believed for some time that NAD- and NADP-GDHs were originated via single gene duplication [57], but as genomes are sequenced and more *gdh* genes are identified this hypothesis has been ruled out. The analysis of phylogenetic distribution patterns of the *gdh* gene families provides strong support for numerous horizontal gene transfer events involving prokaryotes, as well as microbial eukaryotes. Differential gene loss, on the other hand, does not seem to have played an important role in the evolution of *gdh* genes in any of the three domains of life. Sequence comparisons for GDHs from a diverse range of sources show that the hexameric enzymes are similar whatever their coenzyme specificity [58, 59]*.* On the other hand, the tetrameric enzymes are less well understood because of a lower number of characterized tetrameric GDHs. Organisms bearing a tetrameric GDH, which have catabolic roles, also possesses a genetically distinct hexameric NADP-linked enzyme with a biosynthetic role. Mammalian GDHs represent a clear deviation from its ancestral forms, since they have the so-called antenna, a 48 amino acid insertion near the carboxy terminus, although it is not clear when this feature evolved. Sequenced genomes from *Ciliates* show that their GDHs present a smaller *antenna* from that of mammalians, although other members of the Protista, such as trypanosomes, have GDH almost identical to the bacterial forms. *Ciliates* are an evolutionary missing link in the GDH evolution [60]

The structure of GDH of many eukaryotic and prokaryotic organisms has been considerably studied and characterized since the beginning of the 50s. As mentioned above, Most GDHs studied so far are homopolymers consisting of two to six subunits of molecular weight 40,000 to 60,000 (fungal NAD-specific GDHs and bacterial large GDH are exceptions). Most of the characterized GDHs are hexameric and the most common structure found is two trimers of subunits stacked directly on top of each other [61-63]. Some GDHs such as that from bovine liver, which is the best-characterized enzyme [1], may have higher order multimeric structures. This enzyme, which is a hexamer in solution, aggregates to form a high molecular weight species and this polymerization is promoted by a high concentration of enzyme, by high ionic strength and also by allosteric ligands or cofactors [64]. Since the local concentration of GDH in some tissues is very high, aggregation might be a regulatory mechanism of the activity *in vivo*.

Eukaryotic and prokaryotic GDHs share relatively high conservation in their primary and secondary structures [61] and the crystal structures of the bacterial [59, 65, 66] and mammalian forms [61, 63] of GDH confirm that the general architecture and the locations of the catalytically important residues have remained unchanged throughout evolution. Each subunit in this multimeric enzyme is organised into two domains separated by a deep cleft. One domain directs the self-assembly of the molecule into a hexameric oligomer with 32 symmetry. The other domain is structurally similar to the classical pyridine nucleotidebinding domain but with the direction of one of the β-strands reversed. Upon glutamate binding, the enzyme can adopt different conformations by flexing about the cleft between its two domains. NAD+ binds in an extended conformation with the nicotinamide moiety buried deep in the cleft between the two domains [59, 61, 63, 65, 66]. The bottom domains of each trimer make wide contacts with each other, while the NAD+-binding domains bearing the nucleotide-binding motif are poised at the top of the structure.

The largest structural difference between mammalian and bacterial GDH is the *antenna*, which has a helix-loop-helix conformation. The *antenna* ascends from the NAD+-binding domain surface via a long, 23-residue helix and then descends back with a random coil structure. The helices of the "*antenna*" domains in each subunit of the trimer wrap around each other with a right-handed twist to form the core of the antenna protrusion. Extensive contacts between "antennae" may represent hexamer interactions in solution and, perhaps, with other enzymes within the mitochondrial matrix [61]. The fact that *antennae* are only found in the forms of GDH that are allosterically regulated by numerous ligands leads to the interpretation that it plays a major part in this regulation. In contrast to the extensive allosteric homotropic and heterotropic regulation observed in mammalian GDH (see below), bacterial forms of GDH are relatively unregulated.
