Structural and mutagenesis studies of leishmania triosephosphate isomerase: a point mutation can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power

John C. Williams1, Johan P. Zeelen2, Gitte Neubauer, Gert Vriend, Jan Backmann3, Paul A.M. Michels4, Anne-Marie Lambeir5 and Rik K. Wierenga6,7

European Molecular Biology Laboratory, Meyerhofstrasse 1, Postfach 102209, D-69012 Heidelberg, Germany, 3 Vrije Universiteit Brussel, Instituut voor Moleculaire Biologie, Paardenstraat 65, B-1640 Sint-Genesius-Rode, 4 Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry, Catholic University of Louvain, Avenue Hippocrate 74, B-1200 Brussels, 5 Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2620 Wilrijk, Belgium and 6 Department of Biochemistry, University of Oulu, Linnanmaa, FIN-90571 Oulu, Finland


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The dimeric enzyme triosephosphate isomerase (TIM) has a very tight and rigid dimer interface. At this interface a critical hydrogen bond is formed between the main chain oxygen atom of the catalytic residue Lys13 and the completely buried side chain of Gln65 (of the same subunit). The sequence of Leishmania mexicana TIM, closely related to Trypanosoma brucei TIM (68% sequence identity), shows that this highly conserved glutamine has been replaced by a glutamate. Therefore, the 1.8 Å crystal structure of leishmania TIM (at pH 5.9) was determined. The comparison with the structure of trypanosomal TIM shows no rearrangements in the vicinity of Glu65, suggesting that its side chain is protonated and is hydrogen bonded to the main chain oxygen of Lys13. Ionization of this glutamic acid side chain causes a pH-dependent decrease in the thermal stability of leishmania TIM. The presence of this glutamate, also in its protonated state, disrupts to some extent the conserved hydrogen bond network, as seen in all other TIMs. Restoration of the hydrogen bonding network by its mutation to glutamine in the E65Q variant of leishmania TIM results in much higher stability; for example, at pH 7, the apparent melting temperature increases by 26°C (57°C for leishmania TIM to 83°C for the E65Q variant). This mutation does not affect the kinetic properties, showing that even point mutations can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power at the mesophilic temperature.

Keywords: enzymology/leishmania/stability/structure/triosephosphate isomerase


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Triosephosphate isomerase (TIM; EC 5.3.1.1) is a ubiquitous enzyme, which plays a central role in glycolysis by catalysing the interconversion of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (GAP) (Figure 1Go). This reaction is catalysed with very high efficiency, limited by the diffusion of the substrates into and out of the active site (Knowles, 1991Go). In most organisms TIM is a dimeric enzyme of two identical subunits of ~250 residues. The only exceptions known to date are TIM from Pyrococcus woesei, which is a tetramer of four equal subunits of 220 residues each (Kohlhoff et al., 1996Go), and TIM from Thermotoga maritima, which occurs as a tetrameric fusion protein with phosphoglycerate kinase (Schurig et al., 1995Go). Several crystal structures of TIM, with and without active site ligands, are known (Banner et al., 1975Go; Lolis et al., 1990Go; Wierenga et al., 1991Go; Noble et al., 1993aGo; Mande et al., 1994Go; Delboni et al., 1995Go; Velankar et al., 1997Go; Alvarez et al., 1998Go), as well as many amino acid sequences. The stability of the characterized TIMs varies from about 40°C for TIM of the psychrophilic bacterium Vibrio marinus (Alvarez et al., 1998Go) to above 80°C for TIM of thermostable bacteria such as Thermotoga maritima (Beaucamp et al., 1997Go) and Pyrococcus woesei (Kohlhoff et al., 1996Go).



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Fig 1. Triosephosphate isomerase catalyses the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP). Also shown is the covalent structure of the substrate analogue 2-phosphoglycolate (2PG).

 
Leishmania mexicana is a protozoan parasite, belonging to the Trypanosomatidae family. The parasite is transmitted between mammals by bloodsucking sandflies. In the mammalian host, including man, leishmania infects macrophages resulting in a disfiguring disease known as leishmaniasis. The properties of leishmania TIM (LmTIM) have only recently been described (Kohl et al., 1994Go). Its amino acid sequence is most closely related to the sequence of Trypanosoma brucei TIM (68% sequence identity) (Kohl et al., 1994Go). Trypanosomal TIM (TbTIM) has been well characterized and its crystal structure at 1.83 Å resolution has been described (Wierenga et al., 1991Go).

A striking feature of the sequence of leishmania TIM is the presence of a glutamic acid at position 65, whereas in all other known TIM sequences a glutamine is observed at this position, which is at the beginning of loop-3. As discussed previously (Kohl et al., 1994Go), this glutamine is completely buried inside the dimer interface and is involved in an inter subunit hydrogen bonding network. The nitrogen atom of the side chain acts as a hydrogen bond donor to two buried main chain carbonyl oxygen atoms. One hydrogen bond is to the backbone oxygen of Lys13 in loop-1, a conserved active site residue with `unallowed' dihedral angles in each TIM structure, and the other hydrogen bond is to the backbone oxygen of Ala42 of loop-2. The oxygen atom of the glutamine side chain hydrogen bonds to N(Gly76) of the Thr75–Gly76 peptide bond in loop-3 of the other subunit, which are completely conserved residues. This hydrogen bonding network is extended through the interaction of the Thr75–Gly76 backbone oxygen with the side chain of the completely conserved Arg98 of loop-4. Therefore, Gln65 is an intricate part of a highly conserved hydrogen bonding network across the dimer interface which may stabilize the strained conformation of the catalytic Lys13.

The glutamate found at position 65 in LmTIM is expected to be a destabilizing feature, yet there is no evidence from the published data that LmTIM is less stable than TbTIM (Kohl et al., 1994Go). The crystal structure of LmTIM is therefore of interest, because it will show structural rearrangements that might have occurred to accommodate the sequence change. Here we report the crystal structure of leishmania TIM at 1.8 Å resolution and compare its structure with trypanosomal TIM. In addition, we constructed a leishmania E65Q variant and show that this E65Q variant is considerably more thermostable than the wild-type.


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

Site-directed mutagenesis for making the E65Q variant was carried out using a three-step PCR method (Mikaelian and Sergeant, 1992Go) starting from the recombinant LmTIM gene. The method required a forward primer (primer 1), two antisense primers (primer 2 and 3) and the mutagenic oligonucleotide (5'-GTCATCAGCGCTCAGAACGCG-3'; the mutated codon is underlined). Primers 1 and 3 containing the Nde1 and Pst1 restriction sites had already been used for the sequence verification when the gene was cloned (Kohl et al., 1994Go). Primer 2 (5'-CAGTCCTGGCGCTCCGAGTGGCCC-3') was located downstream of the site of mutagenesis but upstream of primer 3. Amplification was carried out in a Hybaid thermal reactor using Vent DNA polymerase. The amplified Nde1–Pst1 fragment containing the mutation was used to replace the corresponding fragment of the wild-type LmTIM gene in the pET3a vector. The presence of the mutation and correct insertion in the wild-type gene were verified.

Overexpression and purification of wild-type LmTIM and the E65Q mutant

Overexpression in Escherichia coli strain BL21(DE3)pLysS was exactly as described previously (Kohl et al., 1994Go). Bacterial cells of 500 ml culture were collected by centrifugation (30 min at 5000 g at 4°C) and frozen at –80°C. The cells were resuspended in 40 ml of lysis buffer [100 mM triethanolamine (TEA), pH 8, 5 mM reduced dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM NaN3, 0.2 mM phenylmethylsulphonyl fluoride (PMSF)] and lysed by sonication. After centrifugation (30 min, 12 000 g, 4°C), most of the overexpressed protein was present in the supernatant. The protein was further purified by ammonium sulphate precipitation at concentrations between 45 and 65%. The 65% pellet was dissolved in 5 ml of 20 mM TEA, pH 8, 1 mM DTT, 1 mM EDTA, 1 mM NaN3 and dialysed overnight against three changes of the same buffer. The protein was loaded on a 25 ml column of CM-Sepharose CL4B, washed with 50 ml of 20 mM TEA, pH 8, 1 mM DTT, 1 mM EDTA, 1 mM NaN3 and eluted with a 100 ml gradient from 0 to 150 mM NaCl in the same buffer. Active fractions were stored at 4°C. The protein appeared as a single band on Coomassie Brilliant Blue-stained 12% SDS–PAGE gels. The specific activity of the purified LmTIM was 3700 units/mg; the specific activity of the E65Q mutant was 5600 units/mg.

Enzyme assays

Activity was determined at 25°C by monitoring the absorbance of NADH at 340 nm for 10 min in a SpectraMAX340 Microtiterplate Reader (Molecular Devices, Sunnyvale, CA). Routinely, the assay mixture (200 or 80 µl) contained 0.47 mM GAP, 0.42 mM NADH, 10 mg/ml glycerol-3-phosphate dehydrogenase and ~2 ng/ml TIM in 100 mM TEA, pH 7.6. To determine the catalytic parameters, the GAP concentration was varied between 0.1 and 1.25 mM. In the reverse direction the reaction mixture contained 0.25–5 mM DHAP, 5 or 10 mM Na2HAsO4, 1 mM NAD, 25 mg/ml glyceraldehyde-3-phosphate dehydrogenase and ~20 ng/ml TIM. For inhibition measurements eight concentrations of the substrate analogue 2-phosphoglycolate (2PG) between 0.025 and 0.4 mM were used and four GAP concentrations (0.15, 0.3, 0.6 and 1.2 mM). The inhibition constant was obtained by fitting the data using Grafit, assuming competitive inhibition.

CD spectroscopy

CD measurements were performed with a Jasco J-715 spectropolarimeter. The loss of secondary structure of LmTIM and the E65Q mutant was observed by recording the CD signal at 222 nm as a function of temperature. During the experiment the temperature of the sample changed at a rate of ~1°C/min between 30 and 90°C. The temperature of the sample was controlled during the measurements by a sensor built into the cuvette holder. The sensor was connected to a Haake N3 circulating bath which adjusted the temperature of the sample. The temperature scale in the final plots represents the temperature actually measured by the sensor in the immediate vicinity of the sample. The protein was always precipitated after the experiments. The apparent melting temperature at the midpoint of the transition (Tm) was obtained by fitting the experimental data points (CD signal versus temperature) with a sigmoidal function.

Mass spectrometry

All mass spectrometric experiments were carried out using an API III triple quadrupole mass spectrometer (Perkin-Elmer Sciex, Thornhill, ON, Canada) equipped with a nanoelectrospray ion source (Wilm and Mann, 1996Go). For the intact molecular weight measurement the final protein concentration was ~10 µM in 50% methanol–40% water–10% formic acid. For peptide sequencing, 10 pmol of LmTIM were digested with trypsin (sequencing grade, Boehringer Mannheim) in 10 µl of 50 mM ammonium hydrogencarbonate at an enzyme-to-substrate ratio of 1:50 at 37°C for 4 h. An aliquot of the resulting digestion mixture was diluted with 50% methanol–45% water–5% formic acid to a final concentration of 1.5 µM and 1 µl was placed in the spraying capillary of the nanoelectrospray ion source. Several of the peptides were fragmented and their sequence determined, including the N-terminal peptide and the peptide containing residue 65 (residues 60–70). In order to determine unambigously the presence of a glutamic acid residue at position 65, the peptide mixture was esterified and the sequence of the modified peptide (60–70) was determined again. The esterification was carried out essentially as described previously (Hunt et al., 1986Go). Briefly, 1 ml of methanol was cooled at -20°C for 10–20 min, followed by the addition of 100 µl of acetyl chloride. The reagent was left at room temperature for 10 min before use. A 2 µl volume of the tryptic digestion mixture was dried down in a vacuum centrifuge and reconstituted with 5 µl of the reaction mixture. After incubation for 45 min at room temperature, the sample was dried down in a vacuum centrifuge. The peptide mixture was dissolved in 90% water–5% methanol–5% formic acid and desalted using a small column (Wilm and Mann, 1996Go). The peptides were eluted directly into the spraying capillary of the nanoelectrospray source for mass spectrometric sequencing.

Crystallization, data collection, structure determination and refinement

For the crystallization experiments the leishmania TIM (11 mg/ml) was in a 10 mM tris(hydroxymethyl)aminomethane (Tris)–HCl buffer (pH 7.5; 25 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM NaN3) containing also 10 mM of the substrate analogue 2PG. Potential crystallization conditions were checked using screening procedures as described previously (Zeelen et al., 1994Go). Well diffracting crystals grow at room temperature in a few weeks in hanging drops using 2 µl of the protein solution and 2 µl well solution containing 20% PEG6000, 1 mM EDTA, 1 mM DTT, 1 mM NaN3 in a 100 mM acetic acid–NaOH buffer, pH 4.5. Before data collection, the crystal was transferred into a 100 mM citric acid–NaOH buffer, pH 5.9 (including 20% PEG6000, 1 mM EDTA, 1 mM DTT, 1 mM NaN3). After a few hours of equilibration in this buffer, the crystal was mounted in a glass X-ray capillary and the data were collected at room temperature with a MAR image plate area detector. The data were processed and merged with the DENZO package (Gewirth et al., 1995Go). The data quality is presented in Table IGo. The structure was solved in a straightforward manner by molecular replacement using AMORE (Navaza, 1994Go). TbTIM was used as the search model. The rotation function and translation function calculations provided straightforward solutions. The correlation coefficient of the correctly oriented and positioned model was 64.9% and the R-factor was 40.3% for data at 2.5 Å resolution. These calculations confirmed that in this crystal form there is only one subunit per asymmetric unit, as the dimer twofold axis of the positioned LmTIM dimer coincides with a crystallograpic twofold axis. Subsequently, loop-6 (Glu167 to Thr179) and loop-7 (Tyr209 to Gln215), known to adopt different conformations in different structures, were removed from the correctly positioned model and electron density maps were calculated, using programs of the CCP4 package (CCP4, 1994Go). These maps, interpreted using O (Jones et al., 1991Go), showed good density for the substrate analogue 2PG as bound in the active site. These maps also showed clear density for loop-6, indicating that this loop had adopted the closed conformation, in agreement with the presence of 2PG in the active site. Subsequently, the missing loop-6 and loop-7 were rebuilt, the 2PG molecule was added to the model and the structure was refined, initially with X-PLOR (Brünger, 1992Go) and finally with TNT (Tronrud, 1992Go), using all data and the bulk solvent correction. Water molecules were added to the model during the final refinement steps. The final refinement statistics are shown in Table IGo.


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Table I. Crystallographic data
 
Structure analysis

The sequences of LmTIM and TbTIM can be aligned without the introduction of insertions or deletions and therefore the same numbering scheme can be used for both sequences. The active site residues for both enzymes are Lys13, His95 and Glu167. For comparative studies the 1.83 Å structure of trypanosomal TIM [Wierenga et al., 1991 (entry 5TIM in the Brookhaven Protein Data Bank (PDB)] was used. The superpositions, using the C{alpha} atoms of the framework ß-strands and {alpha}-helices, were made with the LSQ option in O (Jones et al., 1991Go). The database search for the interactions of aspartate and glutamate side chains was performed with WHAT IF (Vriend, 1990Go). For the sequence analysis, 46 TIM sequences were extracted form the SWISS-PROT database.


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 Materials and methods
 Results
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The crystal structure of LmTIM was refined to a model with good stereochemistry and with a low crystallographic R-factor (Table IGo) at 1.8 Å resolution. The mode of binding of the substrate analogue 2PG in the active site is well defined. The active site loops (loop-6 and loop-7) are in the closed conformation and the side chain of Glu167 has adopted the swing-in conformation (pointing towards the ligand), as seen previously in other liganded structures (Noble et al., 1993bGo). The Ramachandran plot shows one outlier per subunit, which is the catalytic Lys13. This residue has phi/psi values of phi = 50° and psi = –133° as observed in each TIM structure.

Since a glutamate and a glutamine are indistinguishable in the electron density maps at this resolution, mass spectrometry was used to verify the sequence at position 65. The mass of intact LmTIM was determined to be 27 047 (±3) Da, corresponding to the calculated value from the sequence (Kohl et al., 1994Go) without a leading methionine and without any modifications. This was also confirmed by sequencing of the N-terminal peptide. For this the N-terminal tryptic peptide was fragmented and the sequence determined to be SAKPQPIAAANWK, without any modifications. The tryptic peptide containing residue 65 (residues 60–70) was also fragmented and the sequence determined to be Y60VISAENAIAK70. Moreover, the glutamic acid residue at position 65 was verified by methylation and sequencing of the modified peptide.

Glu65 is buried at the dimer interface. As is shown in Figure 2Go, the side chain of Glu65 points towards the active site of its own subunit; it points away from the equivalent Glu65 of the other subunit (the distance between the CD atoms of the equivalent side chains is 17.7 Å). The OE2/OE1 atoms of Glu65 are at a distance of 8 Å from the active site; for example the OE1(Glu65)–NZ(Lys13) distance is 8.4 Å and the OE1(Glu65)–NE2(His95) distance is 8.1 Å.



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Fig. 2. The C{alpha}-trace of the LmTIM dimer. Subunit-1 is in green and subunit-2 in blue. The catalytic residues of subunit-1 are shown (Lys13, in loop-1; His95, in loop-4; and Glu167, in loop-6). The position of 2-phosphoglycolate (2PG) as bound in the active sites of both subunits is depicted. Also shown is Glu65 (in loop-3) of both subunits. This figure was made with GRASP (Nicholls et al., 1991Go).

 
In accordance with the high percentage of sequence identity between the two proteins, the structures of LmTIM and TbTIM are very similar. For example, the r.m.s. difference between the C{alpha} atoms of equivalent framework residues is 0.4 Å and there are only minor structural differences between LmTIM and TbTIM in the vicinity of the unique glutamate at position 65 (Figure 3Go). There is also no difference in the water structure in this region (Figure 4Go). The comparison shows that the carboxylate group of Glu65 has shifted slightly towards the carbonyl of Lys13 and away from the carbonyl of Ala42. This small adjustment allows in fact for a favourable bidentate hydrogen bonding from O(Lys13) with the syn-orbitals of both carboxylate oxygen atoms of Glu65 (the distances from O(Lys13) to these oxygen atoms are 2.72 and 2.97 Å). These syn-orbitals are known to be more basic than the outward-pointing anti-orbitals (Gandour, 1981Go), ensuring optimum hydrogen bonding of the carboxylic group of Glu65 with O(Lys13). In Figure 4Go the distances between the polar atoms interacting with Glu65(LmTIM) are shown and compared with the equivalent distances in TbTIM. The largest distance difference is seen for the OE2(Glu65)–O(Ala42) which has increased by 0.43 Å in LmTIM (all other distance differences are smaller than 0.32 Å). In LmTIM the OE2(Glu65)–O(Ala42) distance is 3.1 Å, whereas the OE2(Glu65)–O(Lys13) distance is 2.7 Å; this indicates that in LmTIM there exists a relatively short van der Waals contact between OE2(Glu65) and O(Ala42) and a good hydrogen bond between OE1(Glu65), OE2(Glu65) and O(Lys13). Such hydrogen bonds are not exceptional; they do occur in other protein structures, e.g. a database survey shows that potential hydrogen bonds between protonated aspartates or glutamates and main chain oxygen atoms are present in other protein structures, but for only less than 1% of the tested aspartate or glutamate side chains (Table IIGo).



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Fig. 3. Stereoview of the superposition of the structure of LmTIM and TbTIM near Glu65 (LmTIM) and Gln65 (TbTIM). Five peptide regions are shown. The stretch including residues Gly76 and Glu77 is from the adjacent subunit. The dotted lines mark the hydrogen bond between O(Ala42) and N(Trp12) and also the hydrogen bonding network extending from O(Lys13). The purple and blue dots mark the positions of the waters near Glu65(LmTIM) and Gln65(TbTIM), respectively. This figure was made with GRASP (Nicholls et al., 1991Go).

 


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Fig. 4. Schematic diagram of the polar interactions of Glu65 (LmTIM). All contacts (within 3.3 Å) of polar atoms of Glu65 with neighbouring polar atoms are shown. The equivalent contacts in TbTIM (PDB entry 5TIM) are listed below the LmTIM distances. The labels Gln65 and NE2 refer to Gln65(TbTIM). The asterisk indicates the residue from the partner subunit.

 

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Table II. Number of occurrences of short contacts (shorter than 2.8 Å) between a main chain oxygen atom and a side chain carboxylate oxygen atoma
 
The identical environment of Glu65(LmTIM) and Gln65(TbTIM) indicates that the replacement of Glu65 by a glutamine will result in additional favourable hydrogen bonding interactions and therefore in greater stability. Consequently, the variant E65Q was made and characterized. The stability of LmTIM and the E65Q variant was measured by recording CD melting curves (Figure 5Go) in the absence and presence of the substrate analogue 2PG at pH 7. In the absence of the substrate analogue 2PG, the apparent Tm is 57°C for LmTIM and 83°C for the E65Q variant. In the presence of 2PG, Tm is ~67°C for the wild-type LmTIM and 90°C for the E65Q variant. The Tm values of LmTIM are in the normal range; they are somewhat higher when compared to TbTIM, e.g. the Tm values for TbTIM are 52°C in the absence and 57°C in the presence of 2PG (Schliebs et al., 1996Go).



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Fig. 5. CD melting curves of LmTIM and the E65Q variant. The protein was dissolved at a concentration of 0.4 mg/ml in 20 mM 3-N-morpholino)propanesulphonic acid (MOPS), pH 7, 1 mM EDTA. Changes in the CD spectrum were recorded at 222 nm as described in Materials and methods. Data are shown for wild-type LmTIM (squares) and the E65Q variant (circles) both in the absence (closed symbols) and in the presence (open symbols) of 1 mM 2PG. Binding of the substrate analogue 2PG causes an increase in the apparent Tm from 57 to 67°C for LmTIM and from 83 to ~90°C for the E65Q variant.

 
As shown in Figure 6Go, the Tm value of the LmTIM is pH dependent. Below pH 6 Tm is >60°C, whereas above pH 8 Tm is <50°C, owing to the deprotonation of Glu65. Consequently, the difference in Tm between LmTIM and the E65Q variant increases from 18°C at pH 5.3 to 32°C at pH 8.8.



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Fig. 6. pH dependence of the apparent Tm for wild-type ({bullet}) and the E65Q-variant ({circ}). Tm is measured by temperature-dependent changes of the CD signal at 222 nm (see Materials and methods). The protein was dissolved at a concentration of 0.44 mg/ml in 10 mM cacodylate (pH 5.3, 6.0 and 6.9) or borate (pH 7.8 and 8.8) buffer containing 100 mM NaF.

 
In order to verify the effect of the E65Q mutation on the catalytic properties, several kinetic parameters were measured. As shown in Table IIIGo, at pH 7.6 there are virtually no differences in the kinetic parameters of LmTIM and the E65Q variant. The measured values are in the normal range, as observed for TIM purified from other organisms (Lambeir et al., 1987Go).


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Table III. Kinetic parameters of LmTIM and the E65Q variant
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The structural and mutational analysis of LmTIM was carried out to investigate the influence of Glu65 in terms of the structure, the stability and the kinetic properties of LmTIM. The structure comparison of LmTIM and TbTIM has shown that the structural contexts of Glu65(LmTIM) and Gln65(TbTIM) are similar, despite the fact that this residue is involved in a highly conserved hydrogen bonding network. The hydrogen bonding scheme of LmTIM, as determined from a crystal soaked at pH 5.9, indicates that the Glu65 side chain is protonated and hydrogen bonded to O(Lys13), an essential catalytic residue (Figure 4Go). The thermodenaturation data (Figure 6Go) show the effect of an ionizable group with a pKa near 7 for lmTIM, which is considerably higher than that for a free glutamic acid side chain, which is known to be near 4.5 (Qasim et al., 1995Go).

Changes in pKa of buried residues due to the environment of the protein are known (Yang and Honig, 1992Go). One example, the V66K variant of staphylococcal nuclease, shows that the pKa of a buried lysine shifts from 10.2 (in the solvent) to below 6.4 (inside the protein) (Stites et al., 1991Go). Another example concerns a buried glutamate in the complex of a mutated OMTKY3-inhibitor with a protease which causes the pKa of the buried glutamate to become 8.7 (Qasim et al., 1995Go).

Although infrequent, the occurrence of a hydrogen bond between a protonated glutamate and a main chain oxygen atom is also observed in other structures (Table IIGo). In at least one other structure, E.coli 3-isopropylmalate dehydrogenase, the interaction occurs at a buried dimer interface residue, as in LmTIM (Wallon et al., 1997Go). Mutagenesis of this residue, Glu256, into a hydrophobic residue (together with a second point mutation) also increases the stability of this enzyme: the apparent Tm for this double mutant, E256I, M259V, is increased by 7°C (Kirino et al., 1994Go).

The presence of the protonated Glu65 is expected to be a destabilizing feature of LmTIM for two reasons. First, at a pH above the pKa of free glutamate (in water), there is a free energy penalty for burying a protonated glutamate in the interior of the protein (Yang and Honig, 1992Go). Second, although the glutamate and glutamine side chains have the same size, the atomic structure near the Glu65 side chain is optimally able to form hydrogen bonding interactions with a glutamine side chain, but not with a protonated glutamate side chain, it remains deficit of one hydrogen bond donor (Figures 3 and 4GoGo). The notion that Glu65 is a destabilizing feature is in fact confirmed by the significant gain in stability of the E65Q variant (Figures 5 and 6GoGo).

Taking TbTIM as the reference structure, one would expect that a change of Gln65 into a glutamate would destabilize the dimer sufficiently so as to favour the formation of monomers. Such destabilization has been shown to occur for the H47N variant of TbTIM (Borchert et al., 1995Go) and for the R-TIM variant of TbTIM (Schliebs et al., 1997Go). In the latter variant the tip of loop-3 is changed by a point mutation (T75R), by which a small, neutral, residue is changed into a bulky, charged residue, thereby interfering with the fit of loop-3 into a pocket near the active site of the partner subunit; consequently, the dimer stability of R-TIM is reduced. In the H47N variant the dimer interface residue His47 is changed into an asparagine; this relatively minor substitution nevertheless reduces the dimer stability and results in a dimer–monomer equilibrium (Borchert et al., 1995Go). However, LmTIM, with the sequence change Q65E, has the same stability as TbTIM. This is distinctly different from the V66K mutant in staphylococcal nuclease where the stability of the mutant has been compromised (Stites et al., 1991Go). Apparently, LmTIM has evolved to overcome this destabilizing feature. Thus, additional compensating stabilizing interactions must be located elsewhere in the structure of LmTIM.

Based on a sequence comparison alone, LmTIM has three more prolines and three fewer glycines than TbTIM. In general, LmTIM has significantly fewer glycines, 16, and more prolines, 10, than the other TIM sequences, which average 21.7 and 8.2, respectively. The ratio of glycines to prolines for LmTIM is 1.60, the second lowest among all TIM sequences with values ranging from 1.55 to 4.80. Considering the denatured state, this difference would give less conformational space in the unfolded state for LmTIM compared with TbTIM favouring the folded state (Shortle, 1996Go).

In terms of structure, this sequence difference can be compared with TbTIM, which is the most closely related TIM in the database. Within a sphere of 6 Å from CD(Glu65) there are no charged side chain atoms (of aspartates, glutamates, histidines, lysines or arginines), either in LmTIM or in TbTIM; there is only one sequence difference within this sphere, which concerns the residue at position 43. This is a serine in TbTIM and a proline in LmTIM. This sequence difference could to some extent compensate for the energetic costs of the glutamine to glutamate change at position 65. First, the O(Ala42) of the Ala42–Pro43 peptide bond is hydrogen bonded in both structures to N(Trp12) of loop-1. The carbonyl oxygen in an X-Pro peptide bond has been suggested to make stronger hydrogen bonds owing to increased electronegativity at the carbonyl caused by the three C–N bonds at the proline nitrogen (Williamson, 1994Go). Second, the side chain of the proline is better suited for a rather hydrophobic pocket when compared with serine. Also, this serine cannot form satisfactory hydrogen bonds in TbTIM. Finally, the serine-to-proline change is not expected to induce strain in the main chain because the phi/psi values at this position are (–92, 171) in each structure, in good agreement with preferred values for prolines (Karplus, 1996Go).

Other sequence differences between LmTIM and TbTIM may also compensate for the destabilizing Q65E sequence difference. These are mapped on to the C{alpha} trace in Figure 7Go. There are three more sequence differences close to the main chain atoms of Glu65. In leishmania TIM there are Ser63, Val91, Ile92, whereas in trypanosomal TIM these residues are alanine, isoleucine and valine, respectively. The last two sequence differences cause minor packing rearrangements. The first sequence difference could contribute to the stability of LmTIM because the extra side chain atom of Ser96 makes a good (buried) hydrogen bond to O(Ala64) and is not involved in short contacts with neighbouring atoms. As can be seen in Figure 7Go, the residues making up the hydrophobic core are mostly unchanged and there are few differences at the dimer interface. In fact, most of the differences are located away from the active site and are solvent exposed (Figure 7Go).



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Fig. 7. Stereoview of the C{alpha}-trace of the LmTIM subunit. Same view as in Figure 2Go. The trace is coloured according to sequence homology between LmTIM and TbTIM as calculated by Clustal W (Thompson et al., 1994Go); dark blue is identical, red is completely different. 2PG marks the position of the substrate analogue. K13 (in loop-1), H95 (in loop-4) and E-167 (in loop-6) are the catalytic residues. E65 (in loop-3) is the glutamate buried at the dimer interface. P43 (in loop-2) is the only sequence difference near the side chain of Glu65. This picture was made with GRASP (Nicholls et al., 1991Go).

 
The additional stability introduced in LmTIM by the E65Q mutation creates a new protein with a thermal stability normally observed for proteins purified from hyperthermophilic organisms, although only one point mutation (per subunit) has been introduced. The gain in stability results in an increase in Tm from 56 to 83°C, at pH 7. To the best of our knowledge, such a large gain in stability by a point mutation has not been observed before. Another example of a large thermal stabilization has been described for iso-1-cytochrome c, where a point mutation increased Tm by 17°C (Das et al., 1989Go). The engineering of hyperstability has also been achieved in a double mutation variant (G8C/N60C) of the neutral protease of Bacillus stearothermophilus, neutral protease (TLPste) (Mansfeld et al., 1997Go). The introduction of an extra SS bridge (between Cys8 and Cys60) increases the apparent Tm by 17°C (in this case the apparent Tm is defined as the temperature at which after 30 min of incubation 50% of the initial activity remains). Six additional point mutations increase the stability even further such that the half-life at 100°C becomes 3 h (Van den Burg et al., 1998Go). Much increased thermal stability has also been found for a double mutant of the activation domain of human procarboxypeptidase (Viguera et al., 1996Go) and for a sixfold mutant of barnase (Serrano et al., 1993Go).

Despite the large gain in thermal stability, the values of the kinetic parameters of the E65Q variant of LmTIM and also of the eightfold mutant of TLPste and the sixfold mutant of barnase have not changed. This is in contradiction to what has been observed for `wild-type' superstable enzymes which have lower catalytic rates at mesophilic temperatures (Wrba et al., 1990Go; Jaenicke et al, 1996Go). It has been suggested that these lower catalytic rates could be due to superstable enzymes being more rigid at mesophilic temperatures (Wrba et al., 1990Go; Jaenicke et al, 1996Go). Apparently, evolutionary pressure to optimize catalytic performance at thermophilic temperature results in a loss of catalytic activity at mesophilic temperature. However, the point mutation studies of LmTIM, TLP-ste and barnase show that mesophilic enzymes can be converted into superstable enzymes without affecting the catalytic properties at mesophilic temperatures. It is therefore predicted that rational design of superstability, without losing catalytic power, can be achieved for many enzymes after a careful analysis of their structures. Such an analysis can suggest generally applicable mutations (which stabilize the folded state with respect to the unfolded state) such as Gly to Ala, Xaa to Pro or the introduction of SS bridges (Van den Burg et al., 1998Go) or mutations which stabilize helices (Villegas et al., 1995Go; Mainfroid et al., 1996Go) or loops (Zhou et al., 1996Go; Blanco et al., 1998Go) or fill up cavities (Lee and Vasmatzis, 1997Go). Such an analysis can also locate specific destabilizing structural features of the wild-type structure which can be corrected by suitable mutagenesis, as is shown here for leishmania TIM.


    Acknowledgments
 
We gratefully acknowledge the help of Dr Veronique Hannaert (ICP, Brussels) with the mutagenesis experiments. The coordinates and structure factors have been deposited at the PDB; the entry code is 1AMK. This work was supported by EU grant BIO4-CT96-0670.


    Notes
 
1 Present address: Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY10032, USA Back

2 Present address: Max Planck Institut für Biophysik,Heinrich Hoffmann Str. 7, D-60528 Frankfurt, Germany Back

7 To whom correspondence should be addressed. E-mail: rik.wierenga{at}oulu.fi Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received July 23, 1998; revised December 7, 1998; accepted December 11, 1998.