Glutamate dehydrogenase isoenzyme 3 (GDH3) of Arabidopsis thaliana is less thermostable than GDH1 and GDH2 isoenzymes

Plant Physiology and Biochemistry 83 (2014) 225e231

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Research article

Glutamate dehydrogenase isoenzyme 3 (GDH3) of Arabidopsis thaliana is less thermostable than GDH1 and GDH2 isoenzymes Laura Marchi a, Eugenia Polverini b, Francesca Degola a, Enrico Baruffini a, Francesco Maria Restivo a, * a b

 di Parma, Parco Area delle Scienze 11/A, 43124 Parma, Italy Dipartimento di Bioscienze, Universita  di Parma, Parco Area delle Scienze 7/A, 43124 Parma, Italy Dipartimento di Fisica e Scienze della Terra, Universita

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 June 2014 Accepted 5 August 2014 Available online 19 August 2014

NAD(H)-glutamate dehydrogenase (GDH; EC 1.4.1.2) is an abundant and ubiquitous enzyme that may exist in different isoenzymic forms. Variation in the composition of the GDH isoenzyme pattern is observed during plant development and specific cell, tissue and organ localization of the different isoforms have been reported. However, the mechanisms involved in the regulation of the isoenzymatic pattern are still obscure. Regulation may be exerted at several levels, i.e. at the level of transcription and translation of the relevant genes, but also when the enzyme is assembled to originate the catalytically active form of the protein. In Arabidopsis thaliana, three genes (GDH1, GDH2 and GDH3) encode three different GDH subunits (b, a and g) that randomly associate to form a complex array of homo- and hetero-hexamers. In order to asses if the different Arabidopsis GDH isoforms may display different structural properties we have investigated their thermal stability. In particular the stability of GDH1 and GDH3 isoenzymes was studied using site-directed mutagenesis in a heterologous yeast expression system. It was established that the carboxyl terminus of the GDH subunit is involved in the stabilization of the oligomeric structure of the enzyme. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Glutamate dehydrogenase Isoenzymes Subunits Thermal stability

1. Introduction In higher plants several genes encoding NAD(H) dependent glutamate dehydrogenase (GDH; EC 1.4.1.2) have been described (Dubois et al., 2003). Plant GDH is an abundant and ubiquitous mitochondrial enzyme that catalyzes the reversible amination of glutamate and the presence of the enzyme in the different organs/tissue/cells is regulated by developmental and nutritional cues that are not yet fully understood. In Arabidopsis thaliana three GDH genes, GDH1, GDH2 and GDH3, encode the b, a and g subunits of the enzyme (Dubois et al., 2003; Pavesi et al., 2000; Restivo, 2004; Turano et al., 1997; Yamada et al., 2003). These three subunits can randomly associate to form distinct hetero- or homohexamers (GDH1 ¼ 6b; GDH2 ¼ 6a; GDH3 ¼ 6g) depending either on the organ examined or the physiological status of the plant (Fontaine et al., 2006; Igarashi et al., 2009; Miyashita and

Abbreviations: GDH, NAD(H)-dependent glutamate dehydrogenase. * Corresponding author. Tel.: þ39 0521 905603; fax: þ39 0521 905604. E-mail addresses: [email protected], [email protected] (F.M. Restivo). http://dx.doi.org/10.1016/j.plaphy.2014.08.003 0981-9428/© 2014 Elsevier Masson SAS. All rights reserved.

Good, 2008; Restivo, 2004; Skopelitis et al., 2007; Turano et al., 1997; Watanabe et al., 2007). In particular, it was shown that the activity of the GDH3 isoform is specifically localized in the roots of Arabidopsis mature plants and in their immature stamens (Fontaine et al., 2013, 2012; Marchi et al., 2013). Moreover, nitrogen starvation could induce both the homo- (6g) and hetero- (6 b/g) isoforms in the roots of young plants hydroponically cultivated (Marchi et al., 2013). From the data of kinetin treatments of plants, it was then concluded that the C/N status and the cytokinin signaling pathways are involved in the process of GDH3 activation in Arabidopsis (Marchi et al., 2013). By the use of single (gdh1 and gdh2) and double (gdh1-2) GDH mutants impaired in the expression of the b and/or a subunit it was previously evidenced that regulation of transcription of the relevant genes is involved in the determination of the isoenzymatic pattern observed in the different organs of A. thaliana (Fontaine et al., 2013). It was also speculated that compensatory mechanisms, involving reciprocal regulation among GDH1, GDH2 and GDH3 modulate the overall GDH activity and determine the specificity of the isoenzyme profile (Fontaine et al., 2013, 2012). However regulation of GDH isoforms activity may also depend on different (i.e. post-transcriptional) regulatory mechanisms. A paradigmatic example is provided by human GDH

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(hGDH) that, as in plants, has an hexameric structure (Shashidharan et al., 1997). The hGDH subunits are encoded by two genes, hGDH1 and hGDH2, giving rise to different isoforms with tissue specific localization and different thermal stability (Shashidharan and Plaitakis, 2014). In addition several GDH isoproteins differing in molecular mass and isoelectric point have been detected in human brain (Plaitakis et al., 2000). hGDH is subject to a complex allosteric regulation by various ligands such as ADP, GTP and L-leucine depending also (but not exclusively) on the presence of a peculiar structure, named “antenna”, not present in plants and bacteria GDH (Li et al., 2012). Moreover a different type of interaction at the interface of hGDH enzyme subunits has been suggested to provide a novel allosteric site for L-leucine resulting in a fine-tuned mechanism to control enzyme activity (see the review reported above and references therein). In plants, no evidence of allosteric regulation by ADP, GTP or leucine has been until now reported. However, in bacteria, whose GDH also is deprived of an antenna like structure, examples of homo- and hetero-tropic allosteric enzyme regulation have been reported [see (Tomita et al., 2010) and references therein]. Interestingly, plant GDH amino acid sequences are more similar to the archaeobacterial counterparts than to the eukaryotic GDH (Pavesi et al., 2000; Syntichaki et al., 1996). Accordingly an increased thermo stability of plant GDHs has been observed when compared to that of lower and higher eukaryotes (Pavesi et al., 2000; Syntichaki et al., 1996). Thus it may not be excluded that plants may have also evolved, in addition to transcriptional control of gene expression, a post-translational mechanism (albeit different from those previously described for hGDH) to regulate GDH enzyme activity. Moreover it has been speculated that GDH may require a fine tuning and rapidly responding mechanism (and hence at the post-translational level) to cope with transitions from stress to post-stress recovery (Limami et al., 2014, 2008). For instance, GDH subunits assembling to form a hexamer and its stability in a specific cell environment may be crucial for enzyme activity and, as such, the possible target for a regulatory step. In this sense, even if a multiplicity of experimental data concerning the activity of various effectors on the modification of GDH isoenzyme pattern have been reported in the literature, the biochemical mechanisms regulating the process of GDH subunits isomerization in plants have been scarcely considered, with few exceptions (Osuji et al., 2003, 1999, 1997), and are not yet fully understood. In the present work the thermal stability of the different Arabidopsis GDH isoforms was investigated in order to determine the possible presence of a structureefunction relationship of the protein.

activity in plants, gdh2 and gdh1-2 double mutants were transferred to N-starvation conditions as previously described (Marchi et al., 2013): seven day old plants were removed from the solid medium and transferred to Petri dishes filled with 10 ml of 1/5 diluted MS medium containing 1% sucrose (adaptation to liquid culture condition) without shaking. After 2 days plants were quickly washed with sterile demineralized water and transferred to the N-starvation medium (1/5 MS salts deprived of KNO3 and NH4NO3) for 5 days. For sample analysis, whole plants (for WT and gdh1 mutant) or excised roots (for gdh2 and gdh1-2 mutants) were collected, blotted dry on filter paper, dipped in liquid N and stored at 80  C. Nicotiana plumbaginifolia seeds belonging to a GDHA silenced mutant line (Fontaine et al., 2006) were processed, germinated and grown in the same in vitro conditions described above for A. thaliana. Protein extracts of this mutant line were used for reconstruction experiments in which the thermostability of A. thaliana and N. plumbaginifolia GDH were compared (see Supplementary Fig. S1)

2. Materials and methods

2.3. GDH mutant protein construction and heterologous expression in yeast

2.2. Protein extraction and in gel GDH activity staining Proteins were extracted from different tissues as described previously (Fontaine et al., 2006; Restivo, 2004). Briefly, 200 mg of plant tissue was homogenized in 200 ml of extraction buffer composed of 100 mM Tricine (pH 8.0), 10 mM MgSO4, 0.2% (v/v) bmercaptoethanol, 0.5 mM PMSF, 40 mM CaCl2, 0.5% (w/v) polyvinylpyrrolidone, 1 mM EDTA and 0.05% (v/v) Triton X-100 in a 1.5 mL Eppendorf tube using a micro-pestle. After two rounds of centrifugation at 15,000  g for 20 min at 4  C, the resulting supernatant (10 ml) was used for GDH in gel activity detection. In gel NAD-GDH activity detection was performed as described by Loulakakis and Roubelakis-Angelakis (1990), with minor modifications. The GDH activity staining solution, containing 100 mM TriseHCl (pH 8.8), 53 mM sodium glutamate, 0.7 mM NAD, 0.03 mM phenazine methosulphate and 0.3 mM Nitro Blue Tetrazolium (NBT) was supplemented with agarose (BioRad, Hercules, CA, USA) at a final concentration of 0.4% (w/v) and poured onto the gel. Enzyme activity staining was performed at 37  C in the dark and stopped by replacing the staining solution with distilled water. Photographs of the gel were taken with a Kodak EDAS120 digital camera (Eastman Kodak Company, Rochester, NY, USA) and the activity of the different GDH isoenzymes was quantified by the 1D image analysis software provided by the manufacturer. Both PAGE and in gel GDH activity detection were performed at least in triplicate with different plants for each experiment. Comparable results were obtained in each replicate.

2.1. Plant material and growth conditions WT, single (gdh1 or gdh2) and double (gdh1-2) GDH mutants of A. thaliana (ecotype Columbia) were grown in vitro conditions. GDH single mutants were obtained from the Arabidopsis Stock Center and have been described elsewhere (Fontaine et al., 2006); double mutants were obtained by crossing single mutants. Prior to sowing, seeds were surface sterilized for 20 min in a diluted bleach solution (final concentration ¼ 0.5% v/v Sodium Hypochlorite) containing 0.1% (v/v) Tween 20. The seeds were then washed three times with sterile demineralized water and stratified for two days in the dark at 4  C. Stratified seeds were germinated in Petri dishes on 1/2 Murashige-Skoog (MS) agar medium; (MS salts; Duchefa; #M0222, Haarlem, The Netherlands) containing 2% (w/v) sucrose and solidified with 0.8% (w/v) agar) and grown for 7 days at 25  C with a 16 h/8 h photoperiod (150 mmol photons m2 s1). To induce GDH3

Total RNA was extracted from in vitro grown plants using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. Two mg of RNA were then reverse-transcribed using the ImProm-II® reverse transcription kit (Promega, Madison, WI, USA) according to the manufacturer's protocol. The cloning site BamHI was added by PCR to the 50 end of the GDH1 and GDH3 cDNAs using specific primers (Supplementary Table S1). Mutant proteins were obtained by introducing the required amino acid substitutions (A411S for GDH1 and I397V, I406L and S411A for GDH3) in GDH1 and GDH3 cDNA sequences by PCR, using specific primers (Supplementary Table S1). The amplification products were cloned into the pGEM-T easy vector (Promega, Madison, WI, USA), sequenced and sub-cloned in the BamHI and EcoRI or SalI sites of the pYEX-BX vector (Clontech Laboratories, Palo Alto, CA, USA). The resulting vectors were then transferred from Escherichia

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coli (DH10B) to yeast (BY474-1; MATa, his3D1 leu2D0 met15D0 ura3D0) cells. For protein expression, yeast cells were grown to late-log phase at 28  C in 0.67% (w/v) yeast nitrogen base medium (YNB) without amino acids (ForMedium™, Hunstanton, UK), supplemented with 2% glucose (w/v) and 1 g l1 of drop-out powder (Kaiser et al., 1994) containing all the amino acids except those used for plasmid maintenance. Yeast proteins were extracted using the same protocol as that used for plant extracts with some modifications. Yeast cells were collected by centrifugation at 3500  g for 20 min and the resulting pellet homogenized using a beads grinder (Amalgamator mod. TAC 200/S, Linea TAC s.r.l., Asti, Italy) and an oscillation frequency of 4200 p/m with 6 successive shaking periods of 10 s each and a 1 s cooling interval in liquid N between each shaking period. After centrifugation at 15,000  g for 20 min at 4  C, the supernatant was immediately used for GDH activity measurements or stored at 80  C.

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by the Swiss-Model structure assessment tools (including PROCHECK). To build the GDH3 model as a hexameric structure, the coordinates were superimposed on those of each subunit comprising the GDH trimer of P. furiosus and then saved in the same structure file. Subsequently, the hexameric structure was obtained by superimposing the trimer on the hexameric structure of GDH of Thermotoga maritima, which is available in the databank (PDB id code 1B26). The energy of the hexameric model was then minimized by means of the GROMACS 4.5.5 software package (Van Der Spoel et al., 2005) and the Gromos96 ffG53A6 force field. The structural analysis was performed both with the VMD (Humphrey et al., 1996) and the Swiss-Pdb Viewer (Guex and Peitsch, 1997) software packages, and by means of the FirstGlance in Jmol web server (http://firstglance.jmol.org). The analysis of the interfaces between the subunits was performed by the Protein Interfaces, Surfaces, and Assemblies service PISA at the European Bioinformatics Institute (http://www.ebi.ac.uk/pdbe/prot_int/pistart.html).

2.4. GDH thermal stability assay 3. Results Protein extracts from plant tissues or yeast cells were incubated in a water bath at different times and at different temperatures. At the end of the incubation period, they were removed from the water bath and immediately stored in ice. After centrifugation at 12,000  g for 5 min at 4  C, the supernatants were loaded on the polyacrylamide gel. Yeast extracts were pretreated for 15 min at 65  C to inactivate endogenous GDH activity and to eliminate nonspecific activity staining. 2.5. Sequence analysis The 27 plant GDH sequences were retrieved from the Plant Genome Database in a BLASTP search [38] using the A. thaliana GDH1 sequence (At5g18170) and aligned with CLUSTALW (http:// www.ebi.ac.uk/Tools/msa/clustalw2/) software. Sequence data can be found in the GenBank/EMBL database under the following accession numbers: GDH1 Actinidia chinensis ABR45723.1; GDH1 Arabidopsis lyrata EFH48065.1; GDH2 A. lyrata EFH47531.1; GDH3 A. lyrata EFH58577.1; GDH1 A. thaliana AED92515.1; GDH2 A. thaliana AED91157.1; GDH3 A. thaliana AEE74011.1; GDH Asparagus officinalis CAA09478.1; GDH Brassica napus BAB62170.1; GDH2 Camellia sinensis ACH97123.2; GDH1 Glycine max CAI53673.1; GDH2 G. max CAI53674.1; GDH1 Lupinus luteus AAV74197.1; GDHB N. plumbaginifolia CAA69601.2; GDHA N. plumbaginifolia CAA69600.1; GDH Nicotiana tabacum CAD12373.1; GDH1 Oryza sativa BAE48296.1; GDH2 O. sativa BAE48298.1; GDH3 O. sativa BAE48297.1; GDH Physcomitrella patens subsp. patens EDQ71532.1; GDH Picea sitchensis ABK25386.1; GDH Populus trichocarpa EEE95085.1; GDH Ricinus communis EEF36917.1; GDH Solanum lycopersicum AAL36888.3; GDH Vitis vinifera CAA60507.1; GDHB V. vinifera ABF57085.1; GDH Zea mays BAA08445.1.

3.1. Thermal stability of the different GDH isoenzymes Since the overall array of GDH isoforms cannot easily be observed in a single electrophoretic run, we take advantage of the use of the different Arabidopsis GDH mutants. In fact by the use of single (gdh1 and gdh2) and double (gdh 1-2) GDH mutants most of the various homo- and hetero isoforms are clearly resolved (Fontaine et al., 2013; Marchi et al., 2013): the most active GDH isoforms, the 6a and 6b homohexamers and the a/b heterohexamers, are readily visible in WT extracts whereas the 6g homohexamer and b/g heterohexamers are better resolved in the double gdh1-2 mutant and in the single gdh2 mutant root extracts, respectively. Consequently, to study the thermal stability of the different GDH isoforms, root protein extracts of WT, gdh1-2 double mutant and gdh2 single mutant were at first incubated for 20 min at different temperatures ranging from 60 to 80  C. The activity of the 6g homohexamer and of the b/g heterohexamers was completely inactivated at 70  C, whereas the a and b homohexamers and the a/ b heterohexamers remained active even after a 20 min treatment at 75  C (Fig. 1). The relative thermal instability of the 6g homohexamer was confirmed when a mix of root protein extracts of gdh1, gdh2 and, gdh1-2 mutants were incubated for up to 20 min at 70  C. The GDH activity of the 6g homohexamer disappeared after 10 min of heat treatment, whereas that of the 6b and 6a homohexamers was still visible after 20 min of incubation (Fig. 2). The thermoresistance of the 6a or the 6b oligomer is not a specific feature of A. thaliana GDH isoforms since the 6b oligomer of N. plumbaginifolia displayed a comparable resistance to thermal denaturation of the Arabidopsis counterpart (Supplementary Fig. S1). 3.2. GDH protein modeling

2.6. Protein modeling The model for the Arabidopsis GDH3 protein structure was built by means of the comparative modeling technique, after a BLASTP search (Altschul et al., 1990) of the GDH3 sequence of Arabidopsis (Uniprot accession number Q9S7A0) versus the Protein Data Bank (PDB). The GDH of the hyperthermophile archaea Pyrococcus furiosus was found to be the best template, with a complete structure that allowed model building up to the residue S411 (44% of residue identity, PDB id code 1GTM). The model was built by means of the Swiss-Model server (Guex and Peitsch, 1997), after a refinement of the sequence alignment using the ClustalW software (Larkin et al., 2007) and the modeling procedure was performed with the largest amount of user intervention. The quality of the model was verified

A sequence comparison of the 27 plant genes encoding GDH found in the plant genome database was performed in order to find a specific amino acid signature in the GDH3 protein sequence. Three amino acid residues (I397, I406 and S411) located in the C-terminal region of the protein were unique to the GDH3 polypeptide of A. thaliana and A. lyrata (Fig. 3). To investigate a possible role for the three amino acid residues in the lower thermal stability of GDH3, structure-function analyses of the GDH oligomers and of the GDH subunit interactions were performed. This was achieved by building a homohexameric model of GDH3 at atomic resolution, by means of the comparative modeling technique. The model was built using as a template the GDH protein structure of the hyperthermophilic organism

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P. furiosus, which is the resolved structure with the amino acid sequence most similar to that of GDH3 (see Materials and methods Section 2.6). Such an approach allowed the modeling of the structure of Arabidopsis GDH3 up to the key residue S411. When looking at the whole hexameric structure of the GDH3 isoenzyme, it can be seen that it is highly symmetric and is formed by the assembly of two equivalent trimers with a three-fold symmetry (ABC and DEF) (Fig. 4A). It would appear that I406 lies at the interface between two subunits of the same trimer (A:C, C:B and B:A), while S411 is at the interface between two subunits, one in each trimer (A:F, C:D and B:E). In contrast, I397 is in the middle of the same helix and not at the interface and thus almost totally buried (Fig. 4A). Therefore, I406 and S411 are the best candidates to explain the lower thermal stability of GDH3, which could be due to weaker interactions between the g subunits. When the amino acid sequence of GDH3 is aligned to that of GDH1 and GDH2 of other higher plants, the I406isoleucine is substituted by a leucine, which is very similar in dimension and hydrophobicity. For this reason it should not substantially influence

Fig. 2. Thermal stability of GDH homohexamers. Extracts of Arabidopsis gdh1, gdh2 and gdh1-2 mutant roots were mixed and incubated for up to 20 min at 70  C; C ¼ untreated control. Following the high temperature treatments, the extracts were subjected to native PAGE followed by NAD-GDH in gel activity staining. The position of the GDH1 (6b), GDH2 (6a) and GDH3 (6g) homohexamers are indicated on left side of the gel.

the interaction among the subunits, unless of small stereochemical changes that could affect the packing with the surrounding hydrophobic residues, obtaining a less favored interaction energy. On the contrary, the polar serine of S411 is substituted in GDH1 and GDH2 by an alanine. The symmetrical interface containing S411 is made by the two bA-strands belonging to two beta-sheets in opposite subunits, that, linked by several salt bridges, form a large flat floor (Fig. 4B). At the extremities of the contact surface, the Cterminal region of the helix containing S411 interacts with the opposite bA-bB loop. The neighborhood of S411, even if it forms some polar interactions, has a prevalent hydrophobic feature, especially in the opposite subunit (Fig. 4B). Therefore, the substitution of serine with the hydrophobic alanine could strengthen the interaction at the interfaces between the two trimers. 3.3. Heterologous expression of chimaeric GDH proteins Chimaeric GDH3 containing the different amino acid substitutions (I397V, I406L and S411A) found in GDH1 and GDH2 was

Fig. 1. Thermal stability of GDH isoenzymes. Protein extracts of Arabidopsis WT and gdh1-2 mutant roots were incubated for 20 min at temperatures, ranging from 60  C to 80  C; C ¼ untreated control. Following the high temperature treatments, the extracts were subjected to native PAGE followed by NAD-GDH in gel activity staining. The position of the GDH1 (6b), GDH2 (6a) and GDH3 (6g) homohexamers and of the a/b and b/g heterohexamers are indicated on left side of the gel.

Fig. 3. The amino acid (aa) consensus sequence for the last 20 amino acid (carboxylterminal) of plant GDH is shown for A) the whole set (n ¼ 27) of sequences. B) the A. thaliana and A. lyrata GDH3 sequences C) the same set of sequences reported in A, excluding those reported in B. Differences among the amino acid sequence of the plant GDH are written with smaller letters as different amino acids at the same position. This figure was created using the weblogo program (weblogo.berkeley.edu).

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yeast strains expressing the WT (GDH1) or a mutated GDH1 (GDH1-S) 6b homohexamer containing the carboxyl-terminal amino acid residue of GDH3, amino acid substitution A411S, were incubated for 30 min at 68  C or 30 min at 75  C, the activity of the chimaeric GDH1-S protein was significantly lower than that of the native GDH1 isoenzyme (Fig. 5B). Thermal inactivation of GDH1-S, when treated at 75  C, was clearly witnessed by the decrease of band intensity and by increased gel mobility and smearing of the relevant isoform. The effect of the inverse substitution of residue 411 in GDH1-S is greater than in GDH3-A, -LA or -VLA, in terms of thermostability. From a structural point of view, it can be useful to analyze the neighborhood of this residue in the two GDH isoforms. It can be observed that the only not conserved amino acid is the number 405, that in WT GDH1 is an isoleucine, while in WT GDH3 is a threonine. In WT GDH1, the I405 form a large hydrophobic cluster with A411, and several other residues both of the same chain (L24, V400, A401, I406, W409, G410, G408) and of the apposing chains (G46, L48, P42, W152) (Fig. 4B, inset). If A411 is mutated in a polar amino acid, namely the serine, it likely destabilizes the cluster both inside its own chain and with the close subunits. This phenomenon should add to the less favorable interaction of S411 at the subunits interface. 4. Discussion

Fig. 4. Model of Arabidopsis GDH3 protein structure. (A) Homoexameric (6g) complex represented with the accessible surface, colored and labeled by subunits. One trimer is made up of the ABC g subunits and the other by the DEF g subunits. The residues I397 is semi-buried and is colored in green; I406 (in cyan) is at the interface of two subunits of the same trimer; S411 (in black) is at the interface of two subunits each in a different trimer. (B) The A and F subunits (in transparent cartoon) and the residues in the 6 Å neighborhood of S411 (in spacefill). The region of the two linked beta-sheet in the opposite subunits is highlighted in opaque mode and the acidic and basic residues forming the salt bridges are colored in red and blue respectively. The amino acid residues of the neighborhood of S411 are shown colored by type: white ¼ hydrophobic, green ¼ polar, blue ¼ basic. Residue S411 is shown in yellow. Inset: Residues present in the 6 Å neighborhood of T405 (in gray) and S411, with the same colors' scheme. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

expressed in a heterologous yeast system. When protein extracts from yeast strains expressing the WT (GDH3) 6g homohexamer or various mutated forms of GDH3 (GDH3-A ¼ S411A, GDH3LA ¼ I406L þ S411A and GDH3-VLA ¼ I397V þ I406L þ S411A) were incubated at 68  C, there was evidence of an increased stability of the chimaeric GDH3 isoenzymes in comparison to that of the WT GDH3 (Fig. 5A). A closer examination of the results witness the prevalent role of the 411A residue in conferring an increased stability to the chimaeric oligomer as compared to the WT GDH3 isoform: we found that GDH3-A, GDH3-LA and GDH3-VLA share similar thermostability (Fig. 5A). This finding is in agreement with what was estimated by the modeling. When protein extracts from

In a number of studies it has been reported that plant GDH proteins display a higher resistance to thermal inactivation when compared to GDH of prokaryotic, fungal or animal origin (Pavesi et al., 2000; Syntichaki et al., 1996). It is also known that plant GDH shares a greater protein sequence similarity with the archaebacteria thermophiles than with other eukaryotic enzymes (Pavesi et al., 2000; Syntichaki et al., 1996). The analysis we have performed on plant extracts of different GDH mutants of A. thaliana confirmed the peculiar thermal stability of plant GDHs. However differences among the various GDH isoforms, that originate from the random assembling of the three GDH subunits, were observed. In particular, the g subunit containing isoforms (homo- or hetero-oligomers) were less stable than the a or b containing isoforms. A theoretical model of the structure of GDH3 protein suggests that the thermostability of GDH depends on trimer/hexamer formation, which can be modified according to the strength of the subunit interactions. If we consider that in GDH isoenzyme proteins the serine at S411 has been mutated to alanine, this could favor local hydrophobic interactions between the subunits, perhaps leading to increased stability. In addition, since the isoleucine at I406 in the Arabidopsis GDH3 g subunit has been substituted in other plant GDH isoenzymes by a similar hydrophobic leucine, it seems that there is a greater contribution of S411 than I406 in changing the subunit interaction pattern. Expression of the WT and mutated forms of GDH1 and GDH3 in a yeast heterologous expression system confirmed that the two homohexamers 6b and 6g exhibited different thermostability (Fig. 5) and reinforced the hypothesis that this difference in stability could be due to the position of the amino acid residue S411 located at the carboxylic terminus of the GDH3 g subunit. Nevertheless, the not complete recovery of thermostability in GDH3 mutants does not exclude that other residues can contribute to the effect. The inverted mutation in GDH1-S, i.e. A411S, has a greater destabilizing effect probably due to the different kind of amino acid at position 405 in the WT GDH1, that is an hydrophobic isoleucine that form with A411 and with other not polar residues a strong hydrophobic cluster that is destabilized with the A411S mutation. This effect is added to the less favored interaction pattern of S411 at the interface with the apposing subunit.

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Fig. 5. Thermal stability of wild type and mutated forms of GDH3 and GDH1 isoenzymes. A) Protein extracts from yeast strains expressing either the Arabidopsis WT GDH3 g subunit or chimaeric g subunits containing the indicated amino acid substitutions (GDH3-A ¼ S411A; GDH3-LA ¼ I406L and S411A; GDH3-VLA ¼ I397V, I406L and S411A) were incubated at 68  C for up to 45 min. B) Protein extracts from two yeast strains expressing either the Arabidopsis WT GDH1 or GDH1-S (containing the amino acid substitution A411S) b-subunit were incubated either at 68  C for 15 min and 30 min, or at 75  C for 30 min. Following the high temperature treatments, the extracts were subjected to native PAGE followed by NAD-GDH in gel activity staining. The intensity of the bands corresponding to in gel GDH activity is indicated below each band and is expressed as a percentage of the control (C); n.d. ¼ not detectable.

Interestingly, human GDH (hGDH), which is encoded by two genes (GLUD1 and GLUD2), has been shown to occur in two different isoenzymatic forms exhibiting an organ or tissue specific localization and a different thermostability (Shashidharan et al., 1997). Moreover the activities of the two hGDH isoenzymes are differentially regulated, by allosteric mechanisms depending on ligand interactions and subunit oligomerisation (Plaitakis et al., 2000). By substituting specific amino acid residues in the hGDH sequences, it has been possible to demonstrate their relevance in determining the allosteric properties and the thermal stability of the enzyme oligomers (Yang et al., 2004; Zaganas et al., 2002). More recently, it has been proposed that one of the hGDH isoenzymes is part of a multi-enzyme complex, called a metabolon (Clegg et al., 2001). The proteineprotein interactions occurring in this complex are likely to induce allosteric modifications of the enzyme activity thus inducing important modifications in cell metabolism (Hutson et al., 2011). It is therefore possible that similar interactions occur in the different GDH isoenzymes in Arabidopsis. Such variable interactions could define a tissue- or organ-specific pattern for the final holoenzyme activity, which also depends on both developmental and environmental conditions. It would be worth testing this hypothesis by comparing the GDH isoenzyme patterns of Arabidopsis ecotypes that display different response to nitrogen availability (Chardon et al., 2010; North et al., 2009). Acknowledgments We gratefully acknowledge Bertrand Hirel and Peter Lea for a critical reading of the first version of the manuscript. We are also indebted to Antonietta Cirasolo and Roberto Silva for their technical help. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.plaphy.2014.08.003. Contribution L.M. carried out the experiments, E.P. performed protein modeling, F.D. and E.B. helped with electrophoresis analysis and GDH cloning in yeast respectively, L.M. and F.M.R. conceived the

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