Contribution of engineered electrostatic interactions to the stability of cytosolic malate dehydrogenase

Francesca Trejo1, Josep Ll. Gelpí1, Albert Ferrer2, Albert Boronat1, Montserrat Busquets1 and Antoni Cortés1,3

1 Departament de Bioquímica i Biologia Molecular, Facultat de Química, Universitat de Barcelona, Martí i Franqués 1 and 2 Departament de Bioquímica i Biologia Molecular, Facultat de Farmàcia, Universitat de Barcelona, Avda. Diagonal 643, E-08028 Barcelona, Spain


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Protein engineering is a promising tool to obtain stable proteins. Comparison between homologous thermophilic and mesophilic enzymes from a given structural family can reveal structural features responsible for the enhanced stability of thermophilic proteins. Structures from pig heart cytosolic and Thermus flavus malate dehydrogenases (cMDH, Tf MDH), two proteins showing a 55% sequence homology, were compared with the aim of increasing cMDH stability using features from the Thermus flavus enzyme. Three potential salt bridges from Tf MDH were selected on the basis of their location in the protein (surface R176-D200, inter-subunit E57–K168 and intrasubunit R149–E275) and implemented on cMDH using site-directed mutagenesis. Mutants containing E275 were not produced in any detectable amount, which shows that the energy penalty of introducing a charge imbalance in a region that was not exposed to solvent was too unfavourable to allow proper folding of the protein. The salt bridge R149–E275, if formed, would not enhance stability enough to overcome this effect. The remaining mutants were expressed and active and no differences from wild-type other than stability were found. Of the mutants assayed, Q57E/L168K led to a stability increase of 0.4 kcal/mol, as determined by either guanidinium chloride denaturalization or thermal inactivation experiments. This results in a 15°C shift in the optimal temperature, thus confirming that the inter-subunit salt bridge initially present in the T.flavus enzyme was formed in the cMDH structure and that the extra energy obtained is transformed into an increase in protein stability. These results indicate that the use of structural features of thermophilic enzymes, revealed by a detailed comparison of three-dimensional structures, is a valid strategy to improve the stability of mesophilic malate dehydrogenases.

Keywords: electrostatic interactions/folding/mesophilic enzymes/protein stability/site-directed mutagenesis/thermophilic enzymes


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A variety of enzymes are being used in several fields of the biotechnology industry and further developments will increase the volume and range of enzymes required. Many enzymes isolated from mesophiles have been used, but they easily denature under the conditions required for industrial purposes (e.g. heat, organic solvents and certain chemicals). Enzymes from thermophiles are generally more stable than those from mesophiles and thermophiles are the traditional source of stable enzymes. However, their use is subject to the availability of enzymes. Protein engineering through site-directed mutagenesis of mesophilic enzymes has become an alternative to the use of thermophilic enzymes. However, an increase in protein stability by structural modification is not straightforward, since protein stability results from the combination of several structural factors, most of which are not completely understood. Protein folding is driven by the hydrophobic properties of non-polar amino acid residues. Strengthening the internal hydrophobicity in a protein by amino acid substitution has been tried in order to improve protein stability (Yutani et al., 1987Go; Kimura et al., 1992Go) and extensive studies on the influence of side chain packing on stability are available (Fersht, 1987Go). On the other hand, the introduction of specific non-covalent interactions such as hydrogen bonding and salt bridges has been the subject of several analyses (Nicholson et al., 1988Go; Erwin et al., 1990Go). These studies showed that whereas hydrogen bonds do not significantly influence protein stability, the number of salt bridges present in proteins isolated from thermophilic organisms is significantly higher (Perutz and Raidt, 1975Go; Walker et al., 1980Go; Vihinen, 1987Go; Nishiyama et al., 1996Go; Tanner et al., 1996Go). Other strategies, such as a decrease in protein flexibility, have also been investigated (Matthews et al., 1987Go).

L-Malate dehydrogenases (MDHs) are good protein models for testing strategies to increase stability. They constitute a well-studied protein family that includes enzymes from both mesophilic and thermophilic organisms whose three-dimensional structure is known. The amino acid residues involved in the enzymatic process have also been characterized. Moreover, MDHs are oligomeric proteins (most of them dimers) and so the analysis can be extended to the dissociation process, which may be a key step in deactivation (Staniforth et al., 1994Go; Ruggia et al., 2001). Few studies involving changes in stability of MDHs are available. Goward et al. reported the increase in Escherichia coli MDH by removing an ionic residue from the active site (Goward et al., 1994Go). However, as the residue involved is also necessary to the protein function, activity is severely compromised. On the other hand, Kono et al. analysed several variants of Thermus flavus MDH (Tf MDH) with changes in side chain hydrophobic packing to test a theoretical prediction of protein stability (Kono et al., 1998Go). The stability of closely related enzymes such as lactate dehydrogenase that share a common fold with MDHs has also been studied (Wigley et al., 1987Go; Nobbs et al., 1994Go). However, none of these studies provides a systematic approach to determine which structural factors determine stability in the MDH framework.

In the present study, we chose pig cytosolic L-malate dehydrogenase (cMDH) as a model. The protein can be obtained in large amounts from a recombinant source (Trejo et al., 1996Go) and easily modified by site-directed mutagenesis. Its three-dimensional structure in several conformations is available (Birktoft et al., 1989Go; Chapman et al., 1999Go). cMDH, which is a mesophilic protein, shows a 55% sequence homology with a thermophilic protein, Tf MDH, whose three-dimensional structure is also known (Kelly et al., 1993Go). The comparison between the two structures can lead to the isolation of structural determinants of the increased stability of Tf MDH. We have analysed the possibility of introducing three potential salt bridges using residues present in the T.flavus enzyme into the cMDH structure. Potential salt bridges have been selected to cover three chemical environments, surface residues, internal residues and inter-subunit contacts without compromising enzyme activity. The analysis of thermal and guanidium chloride denaturation was used to monitor changes in the stability of the modified enzymes.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reagents

Chemicals were purchased from either Sigma Chemical or Roche Molecular Biochemicals and were of the highest grade available. All polymerase chain reactions (PCRs) were achieved with Expand High Fidelity Polymerase from Roche Molecular Biochemicals.

Mutagenesis

In order to optimize the purification process of the mutant enzymes, we first modified the DNA encoding malate dehydrogenase isolated from a pig heart cDNA library (Trejo et al., 1996Go) by inclusion of a C-terminus His-tag (Trejo et al., 1999Go).

Mutants of cMDH were generated by the overlap extension method developed by Horton and Pease (Horton and Pease, 1991Go). All PCRs were carried out with 0.3 µM of each primer (obtained from Amersham-Pharmacia), 0.2 mM of each dNTP (Amersham-Pharmacia) and 2.6 Units of Expand High Fidelity Polymerase in 1x Expand HF buffer containing 1.5 mM MgCl2. After the addition of polymerase, the samples were overlaid with 100 µl of mineral oil and the PCR was programmed in a PTC-100 Programmable Thermal Controller (MJ Research) as follows: 94°C for 2 min, 1 cycle; 94°C for 15 s; appropriate annealing temperature for 30 s; 72°C for 1 min, 25 cycles; 72°C for 7 min, 1 cycle.

To construct the single mutants L168K, N275E and K200D, the modified cMDH-coding gene was used as a template. In turn, double mutants were obtained using the corresponding single mutant as a template. All primers used in PCRs are described in Table IGo. The optimal annealing temperatures for each set of two were calculated in all cases using the program Oligo v.4


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Table I. Mutagenic oligonucleotide primers used for the obtention of mutants L168K, Q57E/L168K, N275E, E149R/N275E, K200D and D176R/K200D
 
After cloning in a pUC vector, all mutations were verified by nucleotide sequencing in a Biosystems Model 373 A apparatus, using both the M13 Universal and Reverse sequencing primers and several internal oligonucleotides (Sanger et al., 1977Go).

Production and purification of recombinant enzymes

As the recombinant enzyme (re-cMDH.His), the mutants were expressed in the pKK223-3 plasmid in Escherichia coli TG2 cells as described elsewhere (Trejo et al., 1996Go). Single colonies of each clone were inoculated in 5 ml of Luria broth supplemented with 100 µg Ap/ml and grown at 37°C overnight. The resulting cultures were used to inoculate 100 ml of the same medium, cultured in turn with shaking until A600 = 1.0 and then induced with 1 mM IPTG. E.coli cells were harvested 20 h post-induction and resuspended in 2 ml of 20 mM sodium phosphate buffer, containing 0.5 M NaCl and 10 mM imidazole. Cells were disrupted at 4°C by sonication, cooled on ice, for four 30 s intervals. The cell lysate was then centrifuged for 30 min at 28 000 g to remove the debris. Like the wild-type, the level of expression of all enzymes was up to 30 mg per litre of the original culture (Table IIGo).


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Table II. Comparison of the production levels and properties of re-cMDH.His and their mutated forms
 
The His-tag added to the cMDH gene allowed the mutant purification by immobilized metal ion affinity chromatography (IMAC), using Ni2+ as immobilized cation and an imidazole gradient between 0.1 and 0.5 M in the elution buffer. The cell extract was loaded on to a 5 ml bed volume column of Hi.trap Chelating Sepharose equilibrated with 20 mM sodium phosphate buffer, containing 0.5 M NaCl and 10 mM imidazole. The column was washed with (i) 25 ml of the equilibrating buffer, (ii) 10 ml of the same buffer containing 100 mM imidazole, (iii) 20 ml containing 300 mM imidazole and (iv) 20 ml containing 500 mM imidazole. cMDH activity was eluted from the column with the buffer containing 300 mM imidazole. This method purifies the enzyme and provides an overall yield of >90% in about 2 h (Trejo et al., 1999Go).

Denaturing polyacrylamide gel electrophoresis (SDS–PAGE) was used as a purity criterion, revealing in all cases a single band by the silver stain procedure (Heukeshoven and Dernick, 1985Go).

Enzyme activity and protein assays

MDH activity was measured by determining the change in absorbance at 340 nm due to NADH oxidation. One unit of enzymatic activity (I.U.) is defined as that catalysing the oxidation of 1 µmol of NADH/min at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 0.15 mM NADH, 0.3 mM oxaloacetate (OAA) and 0.05% sodium azide.

The protein concentration was measured following Warburg and Christian (Warburg and Christian, 1942Go). For purified cMDH mutant preparations, the protein concentration was determined from absorbance at 280 nm using the molar absorptivity coefficient ({varepsilon} = 0.8 ml/mg.cm) calculated from amino acid composition data (Trejo et al., 1996Go).

Determination of kinetic parameters

To obtain the apparent kinetic parameters, the enzyme activity of five replicates was determined at 30°C from –{Delta}A340 nm in 1 cm lightpath cells in 50 mM sodium phosphate buffer, pH 7.4, containing 0.05% sodium azide and 1% bovine serum albumin (BSA) as stabilizing agent. The system {varepsilon} value referred to NADH was 6.25 mM–1 cm–1. Constant values were obtained by fitting the appropriate rate equations to experimental data by non-linear regression (Canela, 1984Go).

Denaturation curve in guanidinium chloride (GdmCl)

The stability of re-cMDH.His and mutant enzymes was determined from the denaturation curve measured in the presence of GdmCl, by monitoring the change in tryptophan fluorescence of each protein (Staniforth et al., 1994Go; Trejo et al., 1996Go). Recombinant enzymes at 0.5 µM were incubated for 3 h at 25°C in 50 mM sodium phosphate buffer, pH 7.4, in various concentrations of GdmCl ranging from 0 to 2 M. The fluorescence intensities were recorded using a Shimadzu RF-5000 spectrofluorimeter (excitation at 280 nm, emission at 340 nm). The conformational free energy in the absence of denaturant ({Delta}Gw,i) and the number of amino acid side chains becoming exposed to the solvent ({Delta}ni) were calculated by fitting the collected data to equations reported elsewhere (Trejo et al., 1996Go).

Determination of NADH dissociation constant

The enhancement in NADH fluorescence emission of the formation of the enzyme–NADH binary complex was used to determine the Kd value for NADH. Concentrated enzyme (100 µM) was continuously added to 5 µM NADH in 50 mM Tris–HCl buffer, pH 8.0, at 25°C. The fluorescence was measured with excitation at 340 nm and emission at 450 nm using a Shimazdu RF-5000 spectrofluorimeter. The titration curves were fitted by a non-linear regression to the corresponding equation (Trejo et al., 1996Go), which corrected for the condition that [NADH]total did not equal [NADH]free.

Thermal stability

The thermal stability of the purified re-cMDH.His and mutants was tested by incubating the enzyme sample (50 nM) in 50 mM sodium phosphate buffer, pH 7.4, containing 0.05% sodium azide at various temperatures for 15 min in sealed vessels. Immediately after chilling the samples in ice, the enzymatic activity was measured.

Thermal inactivation

Irreversible enzyme inactivation was measured in 50 mM sodium phosphate buffer, pH 7.4, containing 0.05% sodium azide, with a protein concentration of 50 nM. Samples were incubated at 55°C in sealed vessels for various time periods. The enzymatic activity was determined immediately after the treatment. The activity data were fitted to the following equation:

((1))

Optimal temperature

Thermal dependence was examined spectrophotometrically under the standard assay conditions at temperatures between 30 and 95°C. Sample activities were determined after addition of 10 µl of purified enzyme solution (70 nM) in 50 mM sodium phosphate buffer, pH 7.4, containing 0.05% sodium azide and 0.5 mg/ml BSA to 990 µl of 0.2 mM NADH and 0.3 mM OAA solution in the same buffer without BSA. The concentration of BSA in the reaction mixture was negligible.

Computer graphics and modelling

To analyse the feasibility of the formation of the salt bridges under study, three-dimensional models of mutants were prepared on the basis of the coordinates of pig cMDH (Berstein et al., 1977Go; Birktoft et al., 1989Go).

With the exception of structures involving changes in residues at the inter-subunit interface, for which a whole dimeric model was used, the structures analysed contained a single subunit of the protein, together with a 5 Å thick fragment of the other subunit. Model building, manipulation and energy optimization were performed using InsightII and Discover (Biosym-MSI) on a Silicon Graphics R8000 workstation.

The models were optimized using energy minimization. In all cases, a 10 Å sphere, surrounding the modified residues, was soaked with a 5 Å thick water layer and energy minimized, keeping the remaining protein atoms fixed to their crystallographic positions. Minimization was performed using the all-atom CVFF force field (Dauber-Osguthorpe et al., 1988Go), with point charges set according to the protonation state of the amino acids at pH 7.0. The dielectric constant was fixed at 2. Minimization consisted of 200 iterations of steepest descent followed by conjugate gradients until the maximum derivative was <0.1 kcal/mol.Å.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Modelling studies

Comparison between Tf MDH and cMDH X-ray structures revealed several electrostatic differences. From these data, we analysed which of these charges from Tf MDH can form salt bridges in cMDH. Among all the possibilities, we decided to introduce three potential salt bridges in the cMDH gene by site-directed mutagenesis: (i) the ion pair Q57E/L168K that forms a salt bridge in the subunit–subunit interface of Tf MDH; (ii) the ion pair E149R/N275E that forms a buried salt bridge between domains within the same subunit in the Thermus flavus enzyme and (iii) the solvent exposed ion pair D176R/K200D that may form a salt bridge although it is not present in T.flavus enzyme. Models of the mutated proteins were built and energy was minimized to check the feasibility of the formation of the salt bridge (Figure 1Go).



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Fig. 1. Structure of the potential salt bridges after energy minimization. (A) D176R/K200D; (B) E149R/N275E; (C) Q57E/L168K.

 
Site-directed mutagenesis of cMDH gene

The oligonucleotides used for constructing the mutants of cMDH are summarized in Table IGo (see Materials and methods). The cMDH mutants obtained by the overlap extension method were cloned in a pUC 18 vector. The double-strand sequencing of the constructions pUCL168K, pUCQ57E/L168K, pUCN275E, pUCE149R/N275E, pUCK200D and pUCD176R/K200D showed that the correct mutations had been incorporated and no additional changes had occurred.

Production and purification of recombinant enzymes

The nucleotide sequences containing the single and the double mutations were subcloned in the expression plasmid pKK223-3 and produced in E.coli strain TG2 (Trejo et al., 1996Go). The production of recombinant enzymes was checked by SDS–PAGE, which revealed in all cases, except for N275E and E149R/N275E mutants, the presence of a major protein band of 36 kDa, corresponding to the molecular mass of re-cMDH.His. The K200D, D176R/K200D, L168K and Q57E/L168K enzymes were expressed at or near wild-type levels (see Table IIGo). Purification was performed by IMAC as described in Materials and methods and the electrophoretic behaviour of the final preparations showed a single band on silver staining (data not shown).

Conformation and catalytic activity

To test whether the mutations introduced any disturbance in the general folding of the subunit, several properties associated with the catalytic activity were determined. The values obtained for KM (OAA), KI (OAA) and kcat showed no significant differences between wild-type and mutated proteins or from reported values for recombinant cMDH without His.tag (Trejo et al., 1996Go).

To the same end, the dissociation constant of the enzyme–NADH complex (Kd) was measured by the enhanced fluorescence of NADH. According to the Kd values (see Table IIGo), none of the mutations significantly altered the coenzyme binding site. In addition, the conservation of the global structure is also supported by the fact that mutant enzymes retained their catalytic activity.

Thermal stability

To compare the thermal stabilities of re-cMDH.His and mutants, the enzymatic activity was determined after 15 min of incubation of the samples at various temperatures (Figure 2Go). The mutated forms K200D and D176R/K200D completely lost their activity after incubation at 55°C. At 60°C, the re-cMDH.His retained only 9.3% of its initial activity and L168K retained 3.5%, whereas the mutant Q57E/L168K showed a residual activity of 26.8%.



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Fig. 2. Thermal stability of re-cMDH.His and their mutated forms. The MDH activity was determined as described in Materials and methods with 0.15 mM NADH and 0.3 mM OAA as substrates using a 50 nM concentration of each enzyme. The residual activity was determined using the activity value at t = 0 as a reference for each temperature assayed. (A) (o) cMDH.His; (•) K200D; ({square}) D176R/K200D. (B) (o) cMDH.His; (•) L168K; ({square}) Q57E/L168K.

 
Optimal temperature

The optimal temperatures of re-cMDH.His and the mutated enzymes were determined as described in Materials and methods. The percentage of activity was plotted versus temperature, assigning the 100% activity value to the optimal temperature (Figure 3Go). Under these conditions, the optimal temperature for re-cMDH.His was 65°C. The mutated forms K200D, D176R/K200D and L168K showed similar values (60, 60 and 65°C, respectively), whereas the optimal temperature for the Q57E/L168K enzyme was 80°C, 15°C higher than the wild-type value.



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Fig. 3. Effect of temperature on the activity of re-cMDH.His and their mutated forms. Assays of MDH activity were conducted at the indicated temperatures under standard conditions. The percentage of activity was determined using the maximum activity value obtained for each enzymatic sample as a reference. (A) (o) cMDH.His; (•) K200D; ({square}) D176R/K200D. (B) (o) cMDH.His; (•) L168K; ({square}) Q57E/L168K.

 
Thermal inactivation

The half life (t1/2) at 55°C for irreversible thermal inactivation of re-cMDH.His and its mutated forms showed several differences. The enzymes K200D and D176R/K200D had t1/2 values lower than those obtained with the wild-type enzyme (see Table IIIGo).


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Table III. Comparison of stability properties of re-cMDH.His and their mutated forms
 
The t1/2 of mutant L168K was very close to that of the re-cMDH.His (10.5 and 13 min, respectively). The main difference in thermostability was that the double mutant protein Q57E/N168K showed a 3-fold increase above wild-type.

Denaturation curve in guanidinium chloride (GdmCl)

We then analysed the difference in conformational stability between the re-cMDH.His and its variants using GdmCl as denaturant and monitoring the changes in tryptophan fluorescence. The shape of the curve for all enzymatic forms assayed agreed with previous reports (Trejo et al., 1996Go). The stability parameters deduced (Table IIIGo) were consistent with those obtained in thermal treatments, although this technique led to less evident differences between mutants.


    Discussion
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 Abstract
 Introduction
 Materials and methods
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 References
 
Electrostatic interactions may account for the increased stability of thermophilic enzymes with respect to their mesophilic counterparts (Perutz and Raidt, 1975Go; Walker et al., 1980Go; Vihinen, 1987Go; Nishiyama et al., 1996Go; Tanner et al., 1996Go). The relevant interactions can be easily identified by comparison of three-dimensional structures or even sequences from thermophilic and mesophilic enzymes. In the present study, we applied this rationale to the comparison between pig cytosolic and T.flavus MDHs. Our aim was to analyse the contribution of several potential electrostatic interactions to the stability of these enzymes and to evaluate the feasibility of the simple transfer of the involved residues in increasing the stability of cMDH.

We chose three sets of electrostatic differences between T.flavus and cytosolic MDHs as the most representative. With the numeration corresponding to the cMDH amino acid sequence reported by Trejo et al. (Trejo et al., 1996Go), E57–K168 and R149–E275 form inter- and intra-subunit salt bridges in the thermophilic structure and are replaced by non-interacting residues in the cMDH structure (Birktoft et al., 1989Go; Kelly et al., 1993Go; Chapman et al., 1999Go). The third example is the pair R176 and D200 in T.flavus, which correspond to D and K in cMDH, respectively. These two residues are fully solvent exposed in either enzyme and, although they are at the proper distance, they do not appear to form a significant salt bridge (Kelly et al., 1993Go). The three double mutants and the three single mutants (L168K, N275E, K200D), used as intermediate steps in the preparation of the former, were analysed. The single mutants would give insight into the effects of introducing a charge imbalance, instead of the full ion pair.

The feasibility of the salt bridge formation was first checked by molecular modelling and energy minimization. As shown in Figure 1Go, under these simulation conditions, all three salt bridges were formed. However, since energy minimization overstresses electrostatic interactions, this result only confirms that the bridge can be formed without a significant distortion of the protein backbone.

As shown in Table IIGo, four of the six proteins prepared were produced at levels comparable to the wild-type enzyme. Two of them, N275E and E149R/N275E, were undetectable. This suggests that at least the N275E mutation was destabilizing enough to preclude the correct folding of the protein. N275E introduced a Glu residue in a position that is not solvent exposed and, hence, energetically unfavourable. The formation of the bridge with R149 would compensate this effect but, according to the lack of expression of the double mutant protein, this did not occur. Examination of the structure obtained after energy minimization indicates that this ion pair requires reorganization of the protein backbone, since the T.flavus enzyme has one deletion at position 276. The result obtained shows that the extra energy obtained from the formation of a salt bridge does not suffice to force this backbone reorganization.

The kinetic and structural properties of the remaining mutated proteins assayed are similar to those of the wild-type enzyme (Table IIGo). This is consistent with the fact that none of the modified residues are involved in the catalytic process or the coenzyme binding site. Besides, the shape of the denaturation curves (not shown) obtained with GdmCl is not modified, thus suggesting that the folding process itself is not affected by the mutations. The slight differences observed in the kcat/Km parameters may be attributed to the changes in stability that can compromise the exact determination of kcat values. The comparison of kinetic properties, enzyme stability and coenzyme affinity between the recombinant enzyme (re-cMDH) and modified malate dehydrogenase (re-cMDH.His) revealed that the engineered His-tag did not alter the overall behaviour of the enzyme (Trejo et al., 1999Go)

The stability of the mutated enzyme was determined by several complementary approaches (see Table IIIGo). The energetic changes involved in denaturation were analysed both from the thermodynamic (GdmCl denaturation) and kinetic (thermal inactivation) points of view. The study of the change in the folding {Delta}G obtained from GdmCl denaturation appears to be the least sensitive of the methods assayed. However, as shown in Figure 4Go, there is a good correlation between these parameters and the change in the kinetic {Delta}G barrier than can be deduced from thermal inactivation assays. Although no quantitative analysis can be performed, as the data correspond to different temperatures, the plot suggests that the interactions under study are present in structures only before both the unfolding transition responsible of the changes in Trp fluorescence and the loss of enzyme activity, i.e. in the fully folded structure. On the other hand, the measurements of thermal stability and optimum activity temperature do not provide direct energetic data but allow us to test the practical significance of the mutation performed under conditions closer to an eventual use of the enzyme.



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Fig. 4. Correlation study between thermodynamic {Delta}{Delta}G (obtained from denaturation curves with GdmCl) and activation {Delta}{Delta}G (obtained from the first-order inactivation constants determined using Equation 1Go).

 
In all the proteins assayed, single mutants are less stable than double mutants, especially in the case of K200D ({Delta}{Delta}G = –0.9 kcal/mol), in which there is electrostatic repulsion between the new D200 and the unmodified D176. Owing to the solvent electrostatic screening, this repulsion is not strong enough to prevent the proper folding of the protein. The mutation D176R partially reverses this loss of stability, but the combination of the two mutations does not improve the stability with respect to the wild-type enzyme. This would confirm that the salt bridge does not form, as in the original T.flavus structure (Kelly et al., 1993Go). The overall loss of stability after the double mutation can be attributed to the poorer ability of Arg residues to interact with water with respect to Lys (Richardson and Richardson, 1989Go).

In the case of L168K, improvement of the stability was expected since a charged residue in a region partially exposed to solvent replaces a hydrophobic one. However, the new K168 is at fairly close distance to K169, R229 and K65, which, in turn, introduce electrostatic repulsion. According to the results, the combination of the two factors slightly decreases stability. The second mutation of the set, Q57E, not only reverses this behaviour, but also produces a significantly more stable protein. The double mutant Q57E/L168K shows a shift in the optimal temperature of more than 15°C, a 2.4-fold increase in half-inactivation time and an increase in thermal stability (see Figure 2Go). This confirms that in this case, the electrostatic bridge has been formed and the extra energy obtained is transformed into an increase in protein stability. We point out that the increase in stability appears in both kinetic and thermodynamic measurements (Figure 4Go). This indicates that the dissociation of the dimeric protein, that would break these salt bridges, occurs in the early stages of the denaturation process. For MDH systems, only one previous reference is available. Goward et al. reported an increase in stability of 0.28 kcal/mol by a R102Q mutation that results from the alteration of a residue located in a mobile loop (Goward et al., 1994Go). However, this mutation also implies a significant decrease in enzyme activity, since R102 is required for substrate binding. The mutant reported in this study, Q57E/L168K, shows higher stabilization and maintains full enzyme activity (see Table IIGo), as it does not involve any residue required for catalysis.

The results obtained confirm that in the case of MDH enzymes, electrostatic interactions can contribute significantly to the stability. A limit case is the N275E mutant, which does not fold in a detectable amount. For the active proteins, {Delta}{Delta}G values range from –0.9 kcal/mol (K200D) to +0.4 kcal/mol (Q57E/L168K). These values correspond to repulsion between two surface-charged residues and the formation of an inter-subunit salt bridge, respectively. The energy values agree with those reported in other systems for exposed or partially exposed salt bridges (Anderson et al., 1990Go; Serrano et al., 1990Go; Dao-pin et al., 1991Go; Zhang et al., 1995Go).

The use of structural features from thermophylic enzymes may be a valid strategy to improve the stability of homologous mesophilic enzymes in this enzymatic system. The effect of the mutations assayed on stability is in agreement with their original structure in the T.flavus enzyme, i.e. only one of the three salt bridges assayed, that present in Tf MDH, forms on the cMDH framework. The energy benefits of a salt bridge do not suffice to reorganize the protein backbone, as for the N175E/E149R double mutant. As a result, a significantly larger region of Tf MDH must be copied into cMDH if there is any local difference between the thermophilic and mesophilic enzymes. Modelling and energy minimization techniques, widely used to check mutant structures before preparation, should be carefully applied, since, as shown in this study, they are valid only to confirm that a given structure is possible, and not to confirm its presence or that it is the most stable energetically.


    Notes
 
3 To whom correspondence should be addressed. E-mail: cortes{at} sun.bq.ub.es Back


    Acknowledgments
 
This work was supported by Grants PB93-0770 and PB96-0990 from the Dirección General de Enseñanza Superior e Investigación Científica (DGESIC). Spain. We thank Robin Rycroft for editorial help.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received January 1, 2001; revised July 19, 2001; accepted July 31, 2001.