**3. GDH enzymology and physiological role**

294 Dehydrogenases

*Peptostreptococcus* 

*Streptomyces* 

GdhB (48 KDa)

evolution [60]

c

**Organism cofactor** 

b The Km depends on the substrate concentration

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

**MW enzyme (KDa)** 

**MW subunit (KDa)** 

*asacharolyticus* NAD 266 49 6 18.4 0.82 6 [54]

*clavuligerus* NAD 1100 179 6 [23] *Streptomyces fradiae* NADP 200 49 4 30.8 1.54 28.6 [55] a NAD+-GDH of *Thermus thermophilus* is a heterohexamer composed by two types of subunits: GdhA (46, 5 KDa) and

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

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

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

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

**subunit number**

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

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

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

> 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

hypothesized than the latter acts specifically in arginine catabolism by converting glutamate, a product of the ammonia-producing arginine succinyl transferase (AST) pathway, into 2-OG, since it is allosterically modulated by arginine (positively) and citrate (negatively) [25]. Similarly, the only active GDH from *Bacillus subtilis* (RocG, NAD+ dependent) appears to be involved in arginine and proline catabolism [46]. On the other hand, despite the catabolic function assigned to NAD+-GDHs, the existence of an NAD+ specific GDH with an unusual biosynthetic role has been reported in the oral bacterium *Capnocytophaga ochraea* [35]. In this case, it was found that only the NAD+-GDH ammonium assimilating activity could be detected in cell free extracts, probably due to the high concentration of ammonium and ammonium precursors that can be found in the gingival crevicular fluid. Interestingly, GDHs have been shown to play a substantial and even predominant role in nitrogen assimilation in conditions of N2 fixation in the Gram positive bacteria *Bacillus macerans* and *Bacillus polymyxa* [44, 45]. Nitrogen fixation only occurs under extreme nitrogen-limiting conditions, when nitrogen from other sources is very scarce. In these conditions nitrogen is always assimilated using the GS/GOGAT pathway, since the Km of GS for ammonium is much lower than that of GDH. This is not the case in *B. macerans* and *B. polymyxa*, as in these organisms the GOGAT activity is much lower than GDH activity in nitrogen-fixing cells. A NAD+-GDH involved in glutamate fermentation has also been described in the anaerobic Gram-positive bacteria *Peptostreptococcus asaccharolyticus* [54]. In this organism, GDH is the first enzyme of the glutamate fermentation via the hydroxyglutarate pathway, and can represent as much as 10% of total protein when grown on glutamate. Very high levels of GDH production has also been reported in some hyperthermophilic archaea like *Pyrococcus furiosus* or some *Thermococcus* strains [5, 6, 71]. These preferentially biosynthetic enzymes represent an exceptionally high percentage of total soluble protein of the cell, in some cases up to 20%, which suggests an important role of these enzymes in these organisms at an extremely high temperature for life.

Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance 297

One characteristic of microbial GDHs is the absence of the *antenna* structure that functions as a heterotropic allosteric site. In agreement with this, the vast majority of microbial GDHs do not appear to have this level of complexity in GDH modulation by purine nucleoside phosphates. However, some microbial GDHs also show homotropic and even heterotropic allosteric control, especially those from the GDH180 family. GDHs from *Psychrobacter* sp. TAD1, *Streptomyces clavuligerus* or *Pseudomonas aeruginosa* show positive cooperativity of substrate binding, a common feature associated with the complex regulation in vertebrate GDHs, but unusual in bacteria [23, 25, 37]. Furthermore, the Gram-positive bacterium *Clostridium symbiosum* displays an apparent negative cooperativity and inhibitory effect of the enzyme cofactor in certain conditions of pH and concentration [49]. On the other hand, heterotropic control, either positive or negative, has been found in an increasingly number of microorganisms. Accordingly, some aminoacids such as L-aspartate or L-arginine are positive allosteric effectors of NAD+-GDH from the psychrophilic bacterium *Janthinobacterium lividum* and *P.aeruginosa* [24, 25], while nucleotides such as ATP or AMP modulate NADP+-GDH from *Salmonella enterica* sv. Typhimurium and NAD+-GDH from *S. clavuligerus* and *Thiobacillus novellus* [23, 40, 43]. In the latter case, AMP has been found to be actually an essential activator for *S. clavuligerus* GDH activity. Conversely, some microbial GDHs have allosteric inhibitors, such as TCA cycle intermediates in the archaeon *Halobacterium halobium*, in *Salmonella enterica* sv Typhimurium, and in *P. aeruginosa* [25, 40, 72], or nucleotides such as ADP in the NAD+-GDH of *Capnocytophaga ochraea* [35]. Finally, the NAD+-GDH from the actinomycete *Mycobacterium smegmatis* is modulated by the small

The diverse roles of bacterial GDH in different organisms provide for a variety of regulatory mechanisms. Here we show a few examples for selected bacteria in which transcriptional

Transcriptional regulation of the *gdhA* gene, encoding NADP-GDH, was first described in the diazotrophic enterobacterium *Klebsiella pneumoniae*, and then in *Escherichia coli* [73]. Transcription of *gdhA* is repressed in both enteric bacteria under nitrogen limitation by the general nitrogen control system (Figure 3,A). This is consistent with the fact that low affinity for ammonium limits the use of GDH for glutamate synthesis at ammonium concentrations below 1 mM. The enterobacterial general nitrogen control (Ntr) system is a very well characterized signal transduction and regulatory network encompassing seven elements: the alternative σ factor σ54, encoded by *rpoN*, the uridylyl transferase-uridylyl removing enzyme GlnD, two PII signal transduction proteins, GlnB and GlnK, the two-component system NtrB-NtrC and the LysR-type transcriptional regulator Nac. Nitrogen status is signalled by the intracellular pools of glutamine (indicative of nitrogen sufficiency), and 2-OG (indicative of nitrogen limitation). These signals are perceived by the PII proteins by means of their

protein kinase GarA [26].

**4. Regulation of bacterial GDH gene expression** 

**4.1. Regulation of GDH synthesis in the enterobacteria** 

regulation of GDH genes has been characterized.

Determination of the GDH enzymatic structure has allowed the elucidation of the mechanisms for allosteric regulation and negative cooperativity. The activity of glutamate dehydrogenase in animals is allosterically regulated by purine nucleoside phosphates and other metabolic intermediates. In brief, GTP and ATP are allosteric inhibitors whereas GDP and ADP are allosteric activators. Hence, a lowering of the energy charge accelerates the oxidation of amino acids. Their *in vivo* regulation may be dependent on the metabolic status of the cell, or in the tissue they are located. The intracellular compartmentalization of the cofactors, and the GDH itself, may also drive the reaction in one or the other direction. In vertebrate cells, GDH appears to be localized primarily in the mitochondrial matrix [1]. The effects of nucleosides on mammalian GDH are complex. For the bovine liver GDH, four binding sites per subunit have been described, being the active site (site I), the adenine nucleotide regulatory site (site II), the guanine nucleotide regulatory site (site III) and the reduced coenzyme regulatory site (site IV). Some substrate and effectors bind just the active site (glutamate, oxoglutarate, ammonia, NADP+) while others are able to bind two different sites (NAD+, ADP, NAD(P)H) [19]

One characteristic of microbial GDHs is the absence of the *antenna* structure that functions as a heterotropic allosteric site. In agreement with this, the vast majority of microbial GDHs do not appear to have this level of complexity in GDH modulation by purine nucleoside phosphates. However, some microbial GDHs also show homotropic and even heterotropic allosteric control, especially those from the GDH180 family. GDHs from *Psychrobacter* sp. TAD1, *Streptomyces clavuligerus* or *Pseudomonas aeruginosa* show positive cooperativity of substrate binding, a common feature associated with the complex regulation in vertebrate GDHs, but unusual in bacteria [23, 25, 37]. Furthermore, the Gram-positive bacterium *Clostridium symbiosum* displays an apparent negative cooperativity and inhibitory effect of the enzyme cofactor in certain conditions of pH and concentration [49]. On the other hand, heterotropic control, either positive or negative, has been found in an increasingly number of microorganisms. Accordingly, some aminoacids such as L-aspartate or L-arginine are positive allosteric effectors of NAD+-GDH from the psychrophilic bacterium *Janthinobacterium lividum* and *P.aeruginosa* [24, 25], while nucleotides such as ATP or AMP modulate NADP+-GDH from *Salmonella enterica* sv. Typhimurium and NAD+-GDH from *S. clavuligerus* and *Thiobacillus novellus* [23, 40, 43]. In the latter case, AMP has been found to be actually an essential activator for *S. clavuligerus* GDH activity. Conversely, some microbial GDHs have allosteric inhibitors, such as TCA cycle intermediates in the archaeon *Halobacterium halobium*, in *Salmonella enterica* sv Typhimurium, and in *P. aeruginosa* [25, 40, 72], or nucleotides such as ADP in the NAD+-GDH of *Capnocytophaga ochraea* [35]. Finally, the NAD+-GDH from the actinomycete *Mycobacterium smegmatis* is modulated by the small protein kinase GarA [26].
