Half-life times of “wild type” and mutant GlcDHs in the presence of different KCl concentrations at 40 ºC (A) and 25 ºC (B).
Extremely halophilic Archaea are found in highly saline environments such as natural salt lakes, saltern pools, the Dead Sea and so on. These microorganisms require between 2.5 and 5.2 M NaCl for optimal growth. They can balance the external concentration by accumulating intracellular KCl to concentrations that can reach and exceed saturation. The biochemical machinery of these microorganisms has, therefore, been adapted in the course of evolution to be able to function at salt concentrations at which most biochemical systems will cease to function. The biochemical and biophysical properties of several halophilic enzymes have been studied in great detail; and, as a general rule, it was found that the halophilic enzymes are stabilized by multimolar concentration of salts. In most cases the salt also stimulates the catalytic activity. This stabilization of halophilic proteins in solvents containing high salt concentrations has been discussed in terms of apparent peculiarities in their composition. Since the first amino acid composition determinations, it has become clear that halophilic enzymes present a higher proportion of acidic over basic residues, an increase in small hydrophobic residues, a decrease in aliphatic residues and lower lysine content than their non-halophilic homologues (Lanyi, 1974; Eisenberg, et al., 1992; Madern et al., 2000). Since then, structural analyses have revealed two significant differences in the characteristics of the surface of the halophilic enzymes that may contribute to their stability in high salt. The first of these is that the excess of acidic residues are predominantly located on the enzyme surface leading to the formation of a hydration shell that protects the enzyme from aggregation in its highly saline environment. The second is that the surface also displays a significant reduction in exposed hydrophobic character, which arises not from a loss of surface exposed hydrophobic residues but from a reduction in surface-exposed lysine residues. Nevertheless, although the number of halophilic protein sequences has increased during the last years, the number of high resolution structures that permit the details of the protein solvent interactions to be seen is limited. The role of the reduction in the surface lysines has been largely ignored (Britton et al., 1998, 2006). Furthermore, in several studies, the authors have concluded that it is the precise structural organization of surface acidic residues that is important in halophilic adaptation. Not only is there an increase in acidic residue content, but these residues form clusters that bind networks of hydrated ions (Richard et al., 2000).
Halophilic archaea are considered a rather homogeneous group of heterotrophic microorganisms predominantly using amino acids as their source of carbon and energy. However, it has been shown that some halophilic archaea are able to use not only amino acids but different metabolites as well, as, for example,
To understand the molecular basis of salt tolerance responsible for halophilic adaptation of proteins, to analyze the coenzyme specificity, and to study the mode of zinc-binding, we have chosen as model enzymes two halophilic dehydrogenase proteins involved in carbon catabolism. They are the glucose dehydrogenase (GlcDH) and isocitrate dehydrogenase (ICDH) from the extremely halophilic Archaea
Haloferax mediterraneiglucose dehydrogenase
GlcDH is the first enzyme of a non-phosphorylated Entner-Doudoroff pathway. It catalyses the reaction:
Haloferax volcaniiisocitrate dehydrogenase
The citric acid cycle enzyme, ICDH (EC 184.108.40.206 and EC 220.127.116.11), catalyses the oxidative decarboxylation of isocitrate (Kay & Weitzman, 1987):
The wild-type enzyme from
2. Materials and methods
2.1. Strains, culture conditions and vectors
Vector pGEM-11Zf(+) (Promega) was used for cloning genes and carrying out some site-directed mutagenesis experiments. The expression vector pET3a was purchased from Novagen.
2.2. Site-directed mutagenesis
Site-directed mutations were introduced into genes cloned in pGEM-11Zf(+) or directly into pET3a expression vector. The synthetic oligonucleotide primers (Applied Biosystems and Bonsai Technology) were designed to contain the desired mutation. Mutant construction was carried out by two different methods. In the first, the gene encoding the halophilic dehydrogenases were cloned into pGEM-11Zf(+) and site-directed mutagenesis was performed using the GeneEditorTM
2.3. Protein preparation
2.4. Glucose dehydrogenase analysis
2.4.1. Kinetic assays and data processing
Initial velocity studies were performed in 20 mM Tris–HCl buffer pH 8.8, containing 2 M NaCl and 25 mM MgCl2. The reaction was monitored by measuring the appearance of NAD(P)H at 340 nm with a Jasco V-530 spectrophotometer. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol NAD(P)H/min under the assay conditions (40 ºC).
The kinetic constants were obtained from at least triplicate measurements of the initial rates at varying concentrations of D-glucose and NAD(P)+. Kinetic data were fitted to the sequential ordered BiBi equation with the program SigmaPlot 9.0.
2.4.2. Effect of EDTA concentration
The samples at different NaCl concentration were incubated with increasing EDTA concentration for 5 min at room temperature. After the incubation, the residual activities of the enzymes were measured in the activity buffer defined previously (Bonete et al., 1996).
2.4.3. Effect of temperature on enzymatic stability and activity
The samples at different NaCl concentration were incubated at various temperatures: 55, 60, 65, 70 and 80 ºC. Aliquots were withdrawn at given times for measurement of residual activity. Furthermore, enzymatic activity was assayed between 25 and 75 ºC at the same conditions described previously.
2.4.4. Effect of salt concentration on enzymatic activity and stability
The enzymatic activity was measured, as previously described, in buffer with KCl or NaCl in the concentration range of 0-4 M. The results are expressed as the percentage of the activity relative to the highest activity obtained.
Salt concentration stability studies were carried out at room temperature and at 40 ºC. Purified preparations of enzyme in 2 M KCl were quickly diluted with 50 mM potassium phosphate buffer pH 7.3 to obtain 0.25 and 0.5 M KCl concentrations. Samples were removed at known time intervals, cooled on ice, and the residual enzymatic activity was then measured. The results are expressed as the percentage of the activity relative to that existing before incubation.
2.4.5. Differential scanning calorimetry (DSC)
DSC experiments were performed using a VP-DSC microcalorimeter (MicroCal). Temperatures from 40 ºC to 90 ºC were scanned at a rate of 60 ºC/h using 50 mM potassium phosphate buffer pH 7.3 containing 1 mM EDTA and 0.5 M or 2.0 M KCl, which also served for baseline measurements. Prior to scanning, all samples of protein and buffer were degassed under vacuum using a ThermoVac unit (MicroCal). The protein concentrations were in the range of 50–80 μM (approximately 4–6 mg/ml). The data were analyzed using ORIGIN software v 7.0.
2.5. Isocitrate dehydrogenase analysis
2.5.1. Sequence alignment
Initial alignment with
Oligonucleotide primers containing the necessary mismatches were used for construction of the mutations: R291S, K343D, Y344I, V350A and Y390P.
2.5.2. Kinetic assays and data processing
The activities of native and mutant ICDHs were determined spectrophotometrically at A340 and 30 °C in 20 mM Tris-HCl buffer pH 8.0, 1 mM EDTA, 10 mM MgCl2 (Tris/EDTA/Mg2+) containing 2 M NaCl, 1 mM D,L-isocitrate (Camacho et al., 1995, 2002), with NADP+ or NAD+ as the coenzyme. One unit of enzyme activity is the reduction of 1 μmol of NADP per min. Initial velocities were determined by monitoring the production of NADPH or NADH at 340 nm in a 1-cm light path, based on a molar extinction coefficient of 6200 M-1 cm-1. Kinetic parameters Km and Vmax were calculated for the NADP+ and NAD+ and isocitrate, depending on the cases, and the turnover number (Kcat) and catalytic efficiency (Kcat/Km) were determined for each of the mutants, by fitting the data to the Eadie–Hofstee equation with the SigmaPlot program (Version 1.02, Jandel Scientific, Erkath, Germany) (Rodriguez-Arnedo et al., 2005).
2.5.3. Modeling ICDH
Native ICDH and the mutant ICDH with all five amino acids substituted (SDIAP mutant) were modeled with the Swiss-Model program on ExPASy Molecular Biology Server (http://swissmodel.expasy. org/) based on sequence homology. The program uses Blast and ExNRL-3D (derived from PDB) database for the search of a potential protein mold. These proteins, previously resolved by X-ray analysis, with more than 20 amino acids in length and more than 25% sequence identity were chosen. The construction of the structural model was done with the Promodll program and the minimization of energy with Gromos96. The program calculates all levels of identity between the sample problem and the sequence pattern, and it calculates the relative standard deviation to the average of the corresponding structures models and control.
3.1. Analysis of acidic surface of
3.1.1. Choice of the halophilic GlcDH mutations
Generally, halophilic enzymes present a characteristic amino acid composition, showing an increase in the content of acidic residues and a decrease in the content of basic residues, particularly lysines. The latter decrease appears to be responsible for a reduction in the proportion of solvent-exposed hydrophobic surface. This role was investigated by site-directed mutagenesis of GlcDH from
The three selected residues are considered as surface acidic residues, and they are located in different regions of the protein surface. Later, the 1.6 Å resolution GlcDH structure revealed that the side-chain carboxyl of D172 is involved in interactions with a cluster of surface water molecules near a bound potassium counter-ion. In contrast, the side-chain carboxyl of D216 forms interactions with surface waters in a region in which no counter-ions can be seen. The side-chain carboxyl of D344 lies on the surface, where it interacts with the solvent but also makes hydrogen bonds to the nearby side-chains of T346 and T347. Moreover, multiple alignments (data not shown) with other GlcDH sequences belonging to the MDR superfamily have shown that the acidic residue D216 from
3.1.2. Site-directed mutagenesis and expression of the mutant proteins
Four mutant enzymes were obtained, the triple mutant and the three corresponding single mutant. The triple mutant GlcDH was created with the GeneEditorTM
The four mutant genes were cloned into the pET3a expression vector, and the resulting constructs were transformed into
The purification of the GlcDH mutants were carried out as described previously. However, after 3–4 days, protein precipitation was observed in the fractions of triple mutant GlcDH whose protein concentration was greater than 1 mg/ml. This problem was solved by decreasing the protein concentration or by reducing the salt concentration through dialysis against the buffer containing 1 M NaCl or KCl. This fact indicates that the halophilic properties of the triple mutant protein have been altered, since the wild-type and single mutant proteins were stable for months under these conditions.
3.1.3. Properties of the mutant enzymes
The kinetic parameters of the mutant proteins were determined and compared to those that had previously been obtained with wild-type GlcDH. Their Km values for NADP+ and glucose are essentially similar and no significant differences in the values for Vmax were detected. These results indicated that the kinetic parameters were not affected by the mutations. It is unlikely, therefore, that the mutations in position 172, 216 and 344 influenced the active site or the integrity of the enzyme. Similar results were obtained when residues on the surface were mutated on malate dehydrogenase from
The dependence of enzymatic activity on the concentration of NaCl is shown in Fig. 2. The triple mutant GlcDH shows its maximum activity in a buffer with 0.50–0.75 M NaCl while the wild-type protein has its maximum activity with 1.5 M NaCl. Furthermore, at low salt concentrations the activity of the triple mutant enzyme is higher than the activity of the wild-type GlcDH. At higher salt concentrations, it is lower than the wild-type protein. With the purpose of determining if the observed behavior in the triple mutant protein is due to the presence of just one mutation or of the three modifications, these experiments were also performed with each single mutant protein. The mutants D172K GlcDH and D216K GlcDH show the same profiles as the triple mutant enzyme. In striking contrast, the behavior of the D344K mutant protein is very similar to the profile obtained with the wild-type GlcDH. These results suggest that the D344K modification does not disturb the halophilic characteristics of GlcDH. Therefore, the behavior of the triple mutant GlcDH in the salt concentrations assayed could be due to the introduction of the mutation D172K and D216K. The profiles obtained using buffers with KCl are very similar.
At optimal salt concentration, the activities of the wild-type and mutant GlcDH proteins are very close. The kinetic parameters are very similar too. Therefore, it appears that the different mutations introduced in GlcDH only influence the dependence of enzymatic activity on the salt concentration. However, in similar studies with the dihydrolipoamide dehydrogenase from
The effects of different salt concentrations on the residual activity of wild-type halophilic GlcDH and the four mutant proteins were measured after incubation at 25 ºC and 40 ºC. In the presence of 2 M KCl, neither wild-type enzyme nor mutant proteins were inactivated at the temperatures assayed. In particular, at salt concentrations above 1 M, the proteins were stable for weeks. As salt concentration increases, the proteins were more stable independent of the temperature. However, at low salt concentrations, small differences were observed in the stability of the proteins. The triple mutant and each single mutant protein appeared to be slightly more stable than the wild-type protein at 0.25 and 0.50 M KCl. The behavior of the proteins at 25 ºC was similar, although a decrease in the temperature implies an increase of the period over which the enzymes are stable. The half-life time (t1/2) for each protein was calculated (Table 1) showing that the mutant protein half-life times, either as a single alteration or altogether, are longer than wild type, both at 25 ºC and 40 ºC. However, there are no significant differences between the triple mutant and the single mutant proteins. All showed similar half-life times under the conditions assayed.
Biocalorimetry experiments were carried out under two different KCl concentrations using a DSC. In the presence of 2 M KCl, wild-type and single mutant GlcDH denaturing temperatures range from 74.6 ºC to 75.9 ºC. However, the triple mutant enzyme shows a lower denaturing temperature, between 73.6 ºC and 73.7 ºC. In other words, the triple mutant enzyme is denatured at slightly lower temperatures than are the wild-type and single mutant GlcDHs in the presence of high salt. At 0.50 M KCl (low salt), the results obtained do not reveal significant data; but the protein denaturing temperatures are lower than those obtained in the presence of high salt, independent of protein type (Fig. 3). This decrease was expected because the halophilic proteins are destabilized in low salt. Consequently the denaturing temperatures of the wild-type and mutant enzymes ranged from 59.8 ºC to 60.7 ºC. There were no significant differences between the temperatures.
The data that we have presented indicate that the halophilic properties of the mutant proteins have been modified. Their enzymatic activity and kinetic parameters have been not affected by the mutations. The triple mutant and the single mutants, D172K GlcDH and D216K GlcDH, have reached their maximum activities at lower salt concentrations than wild-type GlcDH and the D344K mutant. It appears that the D344K substitution has no effect on the salt activity profile. Strikingly, in all the cases the mutant proteins were slightly more stable at low salt concentrations than was the wild-type GlcDH, although they require high salt concentration for maximum stability, like a malate dehydrogenase mutant from
3.2. Analysis of the zinc-binding site of GlcDH from
3.2.1. Choice of the GlcDH mutations
Whilst sequence analysis clearly identifies
In the crystal structure of horse liver alcohol dehydrogenase (HLADH), three protein ligands, C46, H67 and C174 coordinate the catalytic zinc (Eklund et al., 1981). Residues analogous to C46 and H67 are conserved in the vast majority of members of the MDR family, while in some enzymes the analogous residue for C174 is glutamate as in
In order to investigate the mode of zinc binding to the halophilic GlcDH, two mutant enzymes were constructed by site-directed mutagenesis. We replaced the D38 present in the active center of the protein with C or A.
3.2.2. Site-directed mutagenesis and expression of the mutant proteins
Site-directed mutagenesis was carried out to replace the D38 residue by cysteine and alanine in the recombinant GlcDH using GeneEditorTM
The mutant enzymes were refolded and purified as described previously (Pire et al., 2001). In both mutants, the activity was lower than that of the wild-type protein, with the D38A mutant being inactive. This result suggests that D38 is an important residue and that the mutation to A38 leaves the enzyme seriously compromised. With respect to the D38C mutant, the maximum activity observed was approximately 30% of the activity of the wild-type enzyme.
3.2.3. Characterization of D38C GlcDH
The kinetic parameter values for mutant D38C GlcDH were determined and compared with those obtained for wild-type GlcDH (Table 2). KmNADP+ differences are not significant; however, the mutation led to a significant increase of the Km for glucose. Moreover, as the Kcat and Kcat/Kmglucose parameters show, the catalytic efficiency of the mutant protein is less than the catalytic efficiency of wild-type GlcDH. These results indicate that the replacement of D38 to C38 in the GlcDH probably affects not only the catalytic zinc-binding site, but also the active site of the protein. The C38 GlcDH decreases the enzyme’s affinity for glucose and its Vmax relative to the wild-type enzyme. Consequently, the catalytic efficiency of the mutant enzyme is reduced.
The zinc ion in the wild-type enzyme can be removed by EDTA treatment to yield an inactive enzyme (Pire et al., 2000). In order to compare the strength of zinc binding in “wild type” and in the D38C mutant, a similar treatment was carried out. Fig. 5 shows that zinc is more weakly bound in the D38C mutant than in the wild-type enzyme. The EDTA concentration needed to inactivate the enzyme is lower than that needed for the wild-type enzyme, and this inactivation was independent of salt concentration. For the wild-type enzyme, the capacity of EDTA to sequester the zinc is lower in the D38C mutant; and it is salt concentration-dependent. In the three NaCl concentration tested, the enzyme lost approximately 80% of its activity in the presence of 0.25 mM EDTA, and it was completely inactive at concentrations higher than 2 mM. However, in the case of the wild-type GlcDH, the EDTA necessary to sequester zinc atom at 3 M NaCl is higher than at 1 M, so the behavior of this protein is dependent on the salt concentration. At concentrations above 4 mM of the chelating agent, the enzyme is completely inactive, regardless of the NaCl concentration. Therefore, the substitution of D38 by C38 in the protein has weakened the binding of zinc ion. The D residue at position 38 in the halophilic glucose dehydrogenase instead of C, which is commonly found at the analagous postion in other members of the medium chain dehydrogenase family, could represent a halophilic adaptation.
The replacement of D38 by C38 makes the binding of catalytic zinc ion of the halophilic GlcDH very similar to that presented by the thermophilic GlcDHs and other MDR family proteins. In order to clarify if C38 instead of D38 modifies the thermal characteristics of the enzyme at different salt concentrations, the effect of the temperature on enzymatic stability and activity were determined.
Generally at low salt concentration, halophilic proteins are less stable. High temperatures can contribute to their destabilization under these conditions. At high salt concentrations, halophilic proteins are stable; but stability can be perturbed by several factors, such as high temperatures. The thermal inactivation results illustrate that both the wild-type and the D38C mutant proteins show higher thermostability when the concentration of NaCl is raised. However, the D38C GlcDH appears to be slightly more thermostable than “wild type” GlcDH at the NaCl concentration assayed. The half-lives calculated for each protein under the different conditions are shown at Table 3. In general, at temperatures of 60-70 ºC the D38C mutant shows a half-life higher than that of wild-type GlcDH. No reliable comparisons can be made at 80 ºC, as at that temperature total inactivation of the enzyme is achieved in a few seconds. Below 60 ºC the differences between the half-lives are not significant.
The replacement of D38 by C38 appears to have a stabilizing effect on the ability of the protein to withstand high temperatures, producing an enzyme that is marginally more stable at high temperature. However, it is clear that the enzymatic activity of the mutant is lower.
3.3. Alteration of coenzyme specificity in
Hfx. mediterraneiGlcDH and Hfx. volcaniiICDH
18.104.22.168. Mutations for the reversal of coenzyme specificity
The ability of dehydrogenases to discriminate between NAD+ and NADP+ lies in the amino acid sequence of the nucleotide-binding βαβ motif. This βαβ motif is centered around a highly conserved Gly–X–Gly–X–X–Gly sequence (where X is any amino acid) connecting the first β strand to the α helix. The presence of an aspartic residue at the C-terminal end of the second β strand is conserved in NAD+-specific enzymes. In many NADP+-specific enzymes, this residue is replaced by a smaller and neutral residue and complemented by a nearby positively charged residue that forms a positively charged binding pocket for adenosine 2’-phosphate. The three-dimensional structure of the cofactor binding-site of
22.214.171.124. Protein properties
All the reversal coenzyme specificity mutants were expressed as inclusion bodies, and refolding was carried out by rapid dilution in the same way as for the wild-type enzyme (Pire et al., 2001). To assess that the enzymes reached their maximum activity in terms of proper refolding, enzyme activity was measured as a function of time after rapid dilution. The wild-type and mutated enzymes behaved similarly during refolding, although the refolding kinetics of the mutants were slower. Maximum activity was reached after approximately 24 h with the mutated enzymes, whereas the wild-type enzyme achieved maximum activity 2 h after the rapid dilution of solubilized inclusion bodies (Pire et al, 2009).
Once the protein was folded, the purification procedures were identical for the wild-type and mutant enzymes (Pire et al., 2001).
126.96.36.199. Kinetics of “wild type” and coenzyme specificity reversal mutant enzymes
The kinetic constants of the wild-type and mutant forms of GlcDH were determined with both coenzymes, NAD+ and NADP+. The kinetic constants for the enzymes are compared in Table 4 A and B.
The Km value of the wild-type enzyme was 11-fold lower for NADP+ than for NAD+, indicating that the enzyme has a strong preference for NADP+. The single substitution G206D increased the Km 74-fold for NADP+ and decreased Kcat 2-fold, resulting in a 150-fold decrease in the Kcat/Km when using NADP+. This was to be expected as the negative charge of D206 would be likely to repel the adenosine 2’-phosphate of NADP+. This single substitution had a positive effect on catalysis with NAD+. In NAD+-dependent enzymes, an aspartic residue in this position confers specificity towards NAD+ by the bidentate hydrogen bonding with the 2’ and 3’ hydroxyl groups of the adenosine of NAD+. The Km in the presence of NAD+ was similar to that of the “wild type”, but Kcat showed a 2-fold increase. The G206D mutant preferred NAD+ to NADP+, showing a Kcat value with NAD+ similar to that of the wild-type enzyme with NADP+; however, the Kcat/Km ratio was still better in the wild-type enzyme with NADP+.
The single mutant R207I showed an increase of 48 times in Km value with NADP+ when compared with the “wild type”; this again was as expected, considering the role of D207 in the stabilization of the negative charge of the adenosine 2’-phosphate group of NADP+. This increase was accompanied by a decrease in Kcat, which clearly makes the R207I mutant less efficient in catalysis with NADP+. For NAD+ the Km value also increased, but at a ratio of 3 times, much lower than the Km increase with NADP+. The R207I substitution also makes the enzyme less efficient with NAD+, with a decrease of 4 times in Kcat/Km; this substitution also increases the Km for glucose. A similar effect was even more pronounced in the single substitution R208N, in which saturation with glucose cannot be achieved, and attempts to calculate Km and Kcat with both coenzymes led to very high standard deviation values.
The activity of the G206D/R207I double mutant with NADP+ was very low (almost undetectable), and as such the kinetic parameters could not be calculated. However, when the coenzyme NAD+ was incubated with this double mutant, it reached the highest Kcat value, between 1.5 and 2 times higher than the Kcat of the wild-type enzyme with NADP+, and between 3 and 4 times higher than the Kcat of the “wild type” with NAD+. These values indicate that the local rearrangement of the active centre due to the mutations makes catalysis more efficient. The dissociation constant for NAD+ in the double mutant decreased 1.7-fold in comparison with KiNAD+ in the “wild type”, but the KmNAD+ value of NAD+ registered a 2-fold increase. The G206D/R207I/R208N triple substitution produced an inactive enzyme with NADP+, confirming that these two arginines are necessary for NADP+ stabilization. Regarding the kinetic parameters with NAD+, as in the double mutant G206D/R207I, the Km values of both substrates were higher than in the “wild type”; but in the triple mutant the Kcat was also lower, and it was the worst catalyst.
In contrast with our results, in alcohol dehydrogenase from gastric tissues of
Although there are some examples of a coenzyme specificity change from NAD+ to NADP+ with only one mutation, it seems that the specificity change from NADP+ to NAD+ is more difficult to reach with single substitutions. This study shows that G206, R207 and R208 are determinant for coenzyme specificity in
One of the most interesting features of proteins is the fact that they keep in their amino acid sequences a substantial record of their evolutionary histories. Surprisingly, homologous proteins in organisms that diverged billions of years are still similar enough to recognize a correspondence in the organization of conserved and variable regions. They can even be used as markers of the evolutionary process itself. Such comparisons have been performed using protein sequence alignments obtained with different algorithms. The alignments may reveal amino acids with a common origin and/or having similar positions in the corresponding three-dimensional structures of each protein.
Molecular evolution is based on the use of alignments to reconstruct gene trees representing, as closely as possible, the historic process of sequence divergence. This reconstruction requires the development of statistical models able to reproduce the process of mutation, drift and selection.
If one represents the structural alignment of a family of proteins in the form of linear sequence of amino acids, one can see that the spatial correspondence of identical amino acids is reflected in the form of sequence identity. The presence of insertions and deletions of specific parts of structure is revealed in the form of holes or gaps (Gómez-Moreno & Sancho, 2003).
The aim of alignment algorithms for amino acid sequences is the relative structural correspondence of residues. Comparative modeling is the extrapolation of the structure to a new amino acid sequence (model) from a known three-dimensional structure of at least one member (mould) of the same family of proteins. The obtained models contain sufficient information to permit experimental design with an acceptable degree of reliability or to allow structural comparison (Gómez-Moreno & Sancho, 2003).
One of the purposes of such an analysis is to select residues for mutation that may cause changes in some of the biochemical characteristics of the protein, such as the variation in coenzyme specificity.
188.8.131.52. Sequence alignment
The ICDHs belong to an ancient and divergent family of decarboxylating dehydrogenases that includes NAD-isopropylmalate dehydrogenase (IMDH) (Dean & Golding, 1997). “This family of dehydrogenases shares a common protein fold, topologically distinct from other dehydrogenases of known structure that lacks the αβαβ binding motif characteristic of the nucleotide-binding Rossman fold (Rossman et al., 1974; Chen et al, 1995). In ICDHs the adenosine moiety of the coenzyme binds in a pocket constructed from two loops and an α-helix (Hurley et al., 1991), although the latter is substituted by a β-turn in IMDH (Imada et al., 1991; Chen et al., 1995).
Dehydrogenases discriminate between nicotinamide coenzymes through interactions established between the protein and the 2’-phosphate of NADP+ and the 2’- and 3’-hydroxyls of NAD+ (Chen et al., 1995). In the NAD-binding site, the introduction of positively charged residues changes the preference of an NAD-dependent enzyme to neutralize the negatively charged 2’-phosphate of NADP+, as it has been demonstrated with engineered dihydrolipomide and malate dehydrogenases (Bocanegra et al., 1993; Nishiyama et al., 1993).
Specificity is governed by (1) residues that interact directly with the unique 2’-hydroxyl and phosphate groups of NAD+ and NADP+, respectively; (2) more distant residues that modulate the effects of the first group; and (3) remote residues (Hurley et al., 1996). The first group of residues includes L344, Y345, Y391 and R395 (
184.108.40.206. Site-directed mutagenesis
In the halophilic enzyme, the R291S, K343D, Y344I, V350A and Y390P (halophilic ICDH numbering) mutations were selected based on homology. The substitutions were made by site-directed mutagenesis. The changes carried out are positively charged residues, such as Arg and Lys; uncharged amino acids, such as Ser; or negatively charged, such as Asp. Lys is a residue that appears to be conserved in many species, which could mean that its positive charge is crucial for proper catalysis by the enzyme. The first mutant made and characterized was R291S. Arg forms a hydrogen bond with the 2’-phosphate of a NADP+, as can be seen in the
After all five amino acids were changed in
Molecular models of both the native enzyme and the mutants showed no significant changes in secondary structure when they were compared with the model of
The introduced mutations in ICDH from
220.127.116.11. Kinetic characterization of mutants
Five amino acid substitutions introduced into wild-type
Isocitrate specificity changed with the first mutation, but in this case, specificity for isocitrate increased 3- to 10-fold with increasing mutations, suggesting that these mutations favored substrate binding. The maximum value for isocitrate binding occurred when the mutant showed specificity for NAD+ only. Thus, the mutations markedly influenced not only the Km for NAD(P)+, but also the Km for isocitrate. The effect of the mutation on the efficiency for NADP+ or NAD+ was evaluated by incorporating the Km for the substrate. This parameter is called the overall catalytic efficiency and is defined as: (Kcat/(Km IC x Km NAD(P)) (Table 5) (Nishiyama et al., 1993). We speculate that some specific interaction between the substrate and NADP+, which differs from the native substrate–coenzyme complex, is responsible for the decrease in activity. The ratio Kcat/Km is a measure of both enzyme efficiency and the degree to which an enzyme stabilizes the transition state (Dean & Golding, 1997).
One might think that a local conformational change induced by specific binding of NADP+ or NAD+ is responsible for the variation in the behavior of the
The comparison of the sequence with that of the
Site–directed mutagenesis has allowed us to (1) extend the understanding of the molecular basis of salt tolerance for halophilic adaptation, (2) analyze the role of sequence differences between thermophilic and halophilic dehydrogenases involving a ligand to the zinc ion, and (3) identify the residues implicated in coenzyme specificity.
The replacement of aspartic residues by lysine residues on the GlcDH surface have led to a modification of the halophilic properties of the mutant enzymes, D172K and D216K being the most significant mutations (Esclapez et al., 2007).
The mutation of D38, a residue that lies close to the catalytic zinc ion, to C38 or A38 led to a significant reduction in and abolition of activity, respectively. These results suggest that this residue is important in catalysis, either in forming a key aspect of the zinc-binding site or in some other process related with substrate recognition. The replacement of D38 by C38 results in the production of a less efficient enzyme with lower enzymatic activity and catalytic efficiency. Furthermore, this mutant shows slightly more thermostability. Although the D38C GlcDH is less active, it has been crystallized in the presence of several combinations of products and substrates. This fact has allowed us to describe many aspects of the mechanism of the zinc-dependent MDR superfamily (Esclapez et al., 2005; Baker et al., 2009).
Structural analysis of the GlcDH from
The results obtained in our study with the halophilic ICDH and the complete switch of coenzyme specificities in IMDH from T. thermophilus (Imada et al., 1991) and ICDHs from E. coli show that coenzyme specificity in the β-decarboxylating dehydrogenases are principally determined by interactions between the nucleotides and surface amino acid residues lining the binding pockets (Rodriguez-Arnedo et al., 2005).
We thank Dr. Rice, Dr. Baker and Dr. Britton, from The University of Sheffield (UK), for helping us to prepare GlcDH structure figures. This work was supported by Grants from Ministerio de Educación (BIO2002-03179 and BIO2005-08991-C02-01).