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
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Abstract |
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Keywords: electrostatic interactions/folding/mesophilic enzymes/protein stability/site-directed mutagenesis/thermophilic enzymes
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Introduction |
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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., 1994; 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., 1994
). 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., 1998
). The stability of closely related enzymes such as lactate dehydrogenase that share a common fold with MDHs has also been studied (Wigley et al., 1987
; Nobbs et al., 1994
). 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., 1996) and easily modified by site-directed mutagenesis. Its three-dimensional structure in several conformations is available (Birktoft et al., 1989
; Chapman et al., 1999
). 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., 1993
). 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.
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Materials and methods |
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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., 1996) by inclusion of a C-terminus His-tag (Trejo et al., 1999
).
Mutants of cMDH were generated by the overlap extension method developed by Horton and Pease (Horton and Pease, 1991). 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 I. The optimal annealing temperatures for each set of two were calculated in all cases using the program Oligo v.4
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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., 1996). 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 II
).
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Denaturing polyacrylamide gel electrophoresis (SDSPAGE) was used as a purity criterion, revealing in all cases a single band by the silver stain procedure (Heukeshoven and Dernick, 1985).
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, 1942). For purified cMDH mutant preparations, the protein concentration was determined from absorbance at 280 nm using the molar absorptivity coefficient (
= 0.8 ml/mg.cm) calculated from amino acid composition data (Trejo et al., 1996
).
Determination of kinetic parameters
To obtain the apparent kinetic parameters, the enzyme activity of five replicates was determined at 30°C from 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
value referred to NADH was 6.25 mM1 cm1. Constant values were obtained by fitting the appropriate rate equations to experimental data by non-linear regression (Canela, 1984
).
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., 1994; Trejo et al., 1996
). 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 (
Gw,i) and the number of amino acid side chains becoming exposed to the solvent (
ni) were calculated by fitting the collected data to equations reported elsewhere (Trejo et al., 1996
).
Determination of NADH dissociation constant
The enhancement in NADH fluorescence emission of the formation of the enzymeNADH 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 TrisHCl 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., 1996), 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:
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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., 1977; Birktoft et al., 1989
).
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., 1988), 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.
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Results |
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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 subunitsubunit 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 1).
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The oligonucleotides used for constructing the mutants of cMDH are summarized in Table I (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., 1996). The production of recombinant enzymes was checked by SDSPAGE, 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 II
). 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., 1996).
To the same end, the dissociation constant of the enzymeNADH complex (Kd) was measured by the enhanced fluorescence of NADH. According to the Kd values (see Table II), 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 2). 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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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 3). 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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The half life (t) 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 t
values lower than those obtained with the wild-type enzyme (see Table III
).
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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., 1996). The stability parameters deduced (Table III
) were consistent with those obtained in thermal treatments, although this technique led to less evident differences between mutants.
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Discussion |
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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., 1996), E57K168 and R149E275 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., 1989
; Kelly et al., 1993
; Chapman et al., 1999
). 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., 1993
). 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 1, 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 II, 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 II). 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., 1999
)
The stability of the mutated enzyme was determined by several complementary approaches (see Table III). 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
G obtained from GdmCl denaturation appears to be the least sensitive of the methods assayed. However, as shown in Figure 4
, there is a good correlation between these parameters and the change in the kinetic
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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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 2). 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 4
). 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., 1994
). 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 II
), 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, 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., 1990
; Serrano et al., 1990
; Dao-pin et al., 1991
; Zhang et al., 1995
).
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.
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Notes |
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Acknowledgments |
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References |
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Received January 1, 2001; revised July 19, 2001; accepted July 31, 2001.