Analysis of the effect of accumulation of amino acid replacements on activity of 3-isopropylmalate dehydrogenase from Thermus thermophilus

Masako Yasugi1,2, Toshiharu Suzuki1,3,4, Akihiko Yamagishi1,5 and Tairo Oshima1

1 Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan, 2 Institute of Applied Biochemistry, University of Tsukuba, Tennodai 1–1–1, Tsukuba, Ibaraki 305-8572, Japan and 3 Department of Life Science, Tokyo Institute of Technology, Nagatsuta 4259, Yakohama 226-8501, Japan 4 Present address: Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8503, Japan


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
A newly selected cold-adapted mutant 3-isopropylmalate dehydrogenase (IPMDH) from a random mutant library was a double mutant containing the mutations I11V and S92F that were found in cold-adapted mutant IPMDHs previously isolated. To elucidate the effect of each mutation on enzymatic activity, I11V and six multiple mutant IPMDHs were constructed and analyzed. All of the multiple mutant IPMDHs were found to be improved in catalytic activity at moderate temperatures by increasing the kcat with a simultaneous increase of Km for the coenzyme NAD+. kcat was improved by a decrease in the activation enthalpy, {Delta}H!=. The multiple mutants did not show large reduction in thermal stability, and one of them showed enhanced thermal stability. Mutation from I11 to V was revealed to have a stabilizing effect. Mutants showed increased thermal stability when the mutation I11V was combined. This indicates that it is possible to construct mutants with enhanced thermal stability by combining stabilizing mutation. No additivity was observed for the thermodynamic properties of catalytic reaction in the multiple mutant IPMDHs, implying that the structural changes induced by the mutations were interacting with each other. This indicates that careful and detailed tuning is required for enhancing activity in contrast to thermal stability.

Keywords: additive effect/cold adaptation/3-isopropylmalate dehydrogenase/kinetic analysis/thermophilic enzyme/thermal stability


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Homologous enzymes from psychrophilic, mesophilic and thermophilic organisms are found to have high similarity in the sequences and topologies. Generally, thermophilic enzymes retain their activity at high temperatures but show low activity at lower temperatures because their activity is highly temperature-dependent. On the other hand, mesophilic and psychrophilic enzymes which show relatively low thermal stability exhibit high activity at lower temperatures. As reported by Johnston and Goldspink (1975), looking at the specific activity and activation parameters of myofibrillar ATPase from fish whose living environments differ in their average temperature, the high specific activity of psychrophilic enzymes can be explained by an activation free energy below that of their mesophilic and thermophilic counterparts. However, multiple alterations of the amino acid sequence between native enzymes prevent us from correlating the difference of specific characteristics to certain amino acid replacements. In recent years, the experiments which enhance the activity at lower temperatures have been performed using subtilisin, ß-glucosidase, and indole glycerol phosphate isomerase and ornithine carbamoyl transferase (Lebbink et al., 2000Go; Merz et al., 2000Go; Taguchi et al., 2000Go; Roovers et al., 2001Go).

3-Isopropylmalate dehydrogenase (IPMDH), encoded by a leuB gene, catalyzes the oxidative decarboxylation of 3-isopropylmalate (IPM) to 2-oxoisocaproate using NAD+ as a coenzyme, which is involved in leucine biosynthesis. In our previous study some cold-adapted mutant IPMDHs of Thermus thermophilus whose activity was higher than that of the wild-type at 30–40°C were isolated from a random mutant library (Suzuki et al., 2001Go; Yasugi et al., 2001Go). There were two types of cold-adapted mutants. Some mutant IPMDHs (for example, V15I and V126M in Table IGo) showed improved kcat at a low temperature (Suzuki et al., 2001Go). The improvement of kcat was revealed to be caused by the destabilization of the Michaelis complex state (Suzuki et al., 2001Go). In other mutant IPMDHs (for example S92F in Table IGo), Km for NAD+ was improved without any change in kcat. The enhancement of its catalytic efficiency was caused by a stabilized transition state (Yasugi et al., 2001Go). However, no mutant IPMDH isolated showed the stabilized energy levels in both Michaelis complex state and transition state at a low temperature.


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Table I. Kinetic parameters and thermal stability of mutant and wild-type (WT) IPMDHs
 
In this report, we isolated mutant IPMDH I11V+S92F from a randomly mutated plasmid library. This mutant contained amino acid replacements that were found in a previous report. In addition, two mutants I11V+V15I+N237D and I11V+S92F had a common mutation, I11V. To elucidate the effects of each mutation on catalytic activity and thermal stability, we analyzed single mutant I11V and mutants with combinations of mutations I11V, V15I, S92F and V126M. The possibility of constructing the mutant IPMDHs which enhanced catalytic activity at low temperatures more than their parental mutants was tested.

One of the methods of designing functional properties in proteins is to combine mutations each of which makes a small improvement in function (Wells, 1990Go). This approach has been successfully used in stabilizing proteins such as barnase (Serrano et al., 1993Go), phage T4 lysozyme (Matsumura et al., 1989Go) and kanamycin nucleotidyltransferase (Matsumuraet al., 1986Go). This approach has been applied to enhance the catalytic efficiency of human carbonic anhydrase III (HCA III) (Tu et al., 1994Go), triosephosphate isomerase (Blacklow et al., 1991Go) and subtilisin (Carter and Wells, 1988Go). We also observed the accumulative effect of multiple mutation on the thermal stability of IPMDHs (Akanuma et al., 1998Go, 1999Go; Tamakoshi et al., 2001Go). The analysis of the effect of accumulation of each amino acid replacement will help us to understand the relationship between individual mutations and the reaction mechanism.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Isolation and construction of mutant IPMDHs

Isolation of cold-adapted mutant IPMDHs was performed by following the method previously described (Yasugi et al., 2001Go). In brief, mutation was introduced to T.thermophilus leuB gene by error prone PCR. A leuB-deficient Escherichia coli strain, OM17 (Tamakoshi et al., 1995Go), was transformed with the library and selected on leucine free minimum medium plate at 30°C where the wild-type T.thermophilus leuB gene does not support the growth of the E.coli cells.

Site-directed mutagenesis of T.thermophilus IPMDH was performed by the method of Kunkel (1985) using plasmid pNV119 (Tamakoshi et al., 1995Go) as a template with the following synthetic oligonucleotides: 5'-CTCGGTGACCTCCGGACCGACCCCGTCCCCGGG-3' for I11V; 5'-GGCCTCGGTGATCTCCGGTCCGACCCCGTCCCCG-3' for I11V+ V15I; 5'-GGCTTTTCCTTAAGAAAAGGAGACCCGTCTCCGGG-3' for S92F; and 5'-ATGAGGACGTCCATCCC-GCGCGCGATCTCCTCC-3' for V126M (substituted bases are underlined). The base substitutions were verified by sequence analysis. Wild-type and mutant IPMDHs used in this study were overexpressed in E.coli OM17 (Tamakoshi et al., 1995Go) and purified to homogeneity, as previously performed (Yamada et al., 1990Go).

IPMDH activity measurement

The temperature-dependent activity of the wild-type and mutant IPMDHs was determined in 50 mM HEPES buffer, pH 8.0, containing 100 mM KCl, 5 mM MgCl2, 5 mM NAD+, and 0.4 mM threo-DL-IPM as previously reported (Yamada et al., 1990Go). Thermodynamic parameters of the catalytic reaction, i.e. changes in activation free energy ({Delta}G!=), enthalpy ({Delta}H!=) and entropy ({Delta}S!=), and changes in van't Hoff free energy ({Delta}Gm), enthalpy ({Delta}Hm) and entropy ({Delta}Sm) were calculated according to the relationships presented in Feller and Gerday (1997).

Consider the first, second and third mutations that affect the {Delta}G of the wild-type by {Delta}{Delta}G1, {Delta}{Delta}G2 and {Delta}{Delta}G3, respectively, in a multiple mutant, {Delta}G differ from the wild-type by {Delta}{Delta}G1+2(+3). Such free energy changes for two or three single mutants can be related to those of a multiple mutant by the equationGo (Wells, 1990Go)


(1)
where {Delta}GI is positive or negative depending upon whether the interactions between the mutants reduce or enhance the functional properties measured.

By comparing the kcat/Km for the parental mutants and multiple mutants, changes in transition-state energy ({Delta}GT!=) caused by an amino acid replacement(s) were calculated from the equationGo (Wilkinson et al., 1983Go)


(2)
where R is the gas constant and T is the absolute temperature. {Delta}{Delta}Gm represents the change between mutant and wild-type in free energy for Michaelis intermediate formation. {Delta}GT!= represents the change in free energy needed to reach the transition state ([ES–NAD+]!=) from the enzyme complex with substrate and free coenzyme (ES+NAD+).

CD measurements

Circular dichroism (CD) was measured with a Jasco-J-720 Spectropolarimeter in a 1-mm path-length cell as previously performed (Hayashi-Iwasaki et al., 1996Go). Protein sample (0.2 mg/ml) was dissolved in 20 mM phosphate buffer (pH 7.0). Loss of secondary structure of the wild-type and mutant IPMDHs was monitored by recording the CD signal at 222 nm as a function of temperature. The temperature was increased at the rate of 1°C/min.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Selection of a cold-adapted mutant IPMDH

In this study, isolation of cold-adapted mutants of T.thermophilus IPMDH was attempted by following the method previously described (Yasugi et al., 2001Go). Escherichia coli OM17 harboring the wild-type T.thermophilus leuB gene cannot grow under these conditions. Five mutant strains that grew on leucine-free minimum medium plate were isolated at 30°C from the mutated plasmid library of 3.2x103 transformants. Nucleotide sequencing of one of the isolated leuB genes revealed two amino acid substitutions, Ile11 to Val (I11V) and Ser92 to Phe (S92F). Both amino acid substitutions have been found in the cold-adapted mutant IPMDHs isolated previously (Suzuki et al., 2001Go). The other four strains had no base replacements in the leuB gene. Mutations may have occurred in the other region of the plasmid or in the host genome.

Preparation of I11V and multiple mutant IPMDHs

To analyze the effects of multiple mutation, several multiple mutants with different combinations of the four mutations were constructed. Mutant leuB gene for I11V and six multiple mutant IPMDHs (I11V+V15I, I11V+S92F, V15I+S92F, V15I+V126M, S92F+V126M and I11V+V15I+S92F) were constructed. Each mutant IPMDH was overexpressed in E.coli cells and purified to homogeneity as judged by SDS–PAGE (data not shown).

Temperature dependence of the specific activity of the mutants

The specific activity of the wild-type and mutant IPMDHs were determined at 30–90°C (Figure 1Go). All the six multiple mutants investigated showed increased specific activity at 40°C. Although a mutant IPMDH with only the I11V mutation was found to have no significant change in specific activity when compared to the wild-type enzyme, the specific activity was enhanced 6.0–8.2-fold at 30°C by adding other amino acid substitutions (I11V+V15I, I11V+S92F, I11V+V15I+S92F). The specific activity of S92F+V126M, and of I11V+ V15I+S92F was slightly higher than that of their parental mutants at lower temperatures (Figure 1D and EGo). This enhancement of specific activity disappeared at higher temperatures.



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Fig. 1. Temperature dependencies of specific activity of wild-type and mutant IPMDHs. •, Wild-type; x, I11V; {circ}, V15I; {triangleup}, S92F; {square}, V126M; {blacktriangleup}, I11V+V15I; |, I11V+S92F; {blacktriangledown}, V15I+S92F; {triangledown}, V15I+V126M; {blacklozenge}, S92F+V126M; {blacksquare}, I11V+V15I+S92F. Specific activity of the wild-type and multiple mutant IPMDHs in units/µg at 40°C was: wild-type, 4.0; I11V, 5.8; V15I, 28.0; S92F, 6.0; V126M, 22.0; I11V+V15I, 22.0; I11V+S92F, 18.7; V15I+S92F, 16.2; V15I+V126M, 16.2; S92F+V126M, 29.2; I11V+V15I+S92F, 28.0.

 
Kinetic and thermodynamic analyses of the catalytic activity

The kinetic parameters of the wild-type and mutant IPMDHs were determined at 40 and 60°C (Table IGo). In V15I+V126M and S92F+V126M mutants, the Km for NAD+ showed intermediate values between those of the parental mutants, while the other multiple mutants showed higher Km than the parental mutants, at 40°C. The kcat of I11V+S92F was higher, while the kcat of the other was either intermediate between or lower than the corresponding factors of their parental mutant IPMDHs at 40°C. However, no kcat/Km-improved mutant was obtained by the combination of mutations. The mutant I11V+V15I+V126M showed an extremely large Km(>75 000 µM) and was omitted from further analysis.

Thermodynamic parameters were estimated from the kinetic parameters (Table IIGo). In all of the multiple mutant IPMDHs, the free energy change of NAD+-binding ({Delta}Gm) increased and the free energy change of activation ({Delta}G!=) decreased when compared to the wild-type. All of these changes were induced by enthalpy change except in the case of I11V+S92F and I11V+V15I+S92F in which the change in {Delta}Gm were induced by entropy change.


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Table II. Thermodynamic parameters (kcal/mol) of catalytic reaction of mutant and wild-type (WT) IPMDHs (40°C)
 
Analysis of thermal stability

The thermal stability of the mutant IPMDHs was assessed by examining CD melting profiles at 222 nm. The apparent melting temperatures, the midpoints of denaturation (Tm), are summarized in Table IGo. I11V and I11V+S92F mutant IPMDHs showed 4°C higher thermal stability than the wild-type. The thermal stability of the other mutants was the same or a little lower than that of the wild-type. Thermal stability of multiple mutants that contain I11V was higher than that of parental mutant other than I11V.


    Discussion
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Temperature dependence of the catalytic activity of mutant IPMDHs

In all multiple mutant IPMDHs, specific activity was higher than that of the wild-type enzyme at moderate temperatures, while the maximal performance at the optimum temperature was lower than that of the wild-type because of its large Km. Especially in V15I+V126M, temperature dependence of specific activity was very low (Figure 1CGo). The lower temperature dependence of specific activity was often observed in other cold-adapted mutant enzymes (Kano et al., 1997Go; Taguchi et al., 1998Go; Suzuki et al., 2001Go; Yasugi et al., 2001Go) and natural psychrophilic enzymes (Huey and Hertz, 1984Go; Huey and Kingsolver, 1993Go; Fields and Somero, 1998Go; Thomas and Cavicchioli, 2000Go). The enzyme with low temperature dependence can avoid reduction of the rate of catalytic reaction caused by temperature change. In this ‘stress-resistant’ mutant, maximum activity is reduced. The range of temperature with substantial activity and the maximum activity may be inversely related. Huey and co-workers have expressed the characteristics ‘jack-of-all-temperature is a master of none’ (Huey and Hertz, 1984Go; Huey and Kingsolver, 1993Go). The reduction of temperature dependence of catalytic activity is one of the characteristics of cold adaptation mechanisms well observed in ectothermic organisms.

The multiple mutant IPMDHs were found to be cold-adapted mutants which improved their activity compared to the wild-type at lower temperatures by increasing their kcat (Table IGo). Increase in kcat in multiple mutants was accompanied by the increase in Km for NAD+ compared to the wild-type (Figure 2Go). This simultaneous increase in kcat and Km was also seen in cold-adapted mutants of T.thermophilus IPMDH (Suzuki et al., 2001Go; Yasugi et al., 2001Go), and natural psychrophilic enzymes A4-LDH (Fields and Somero, 1998Go) and elastase (Asgeirsson and Bjarnason, 1993Go). Enhancement of the catalytic activity at low temperatures has been explained by the reduced affinity for NAD+ in kcat-improved cold-adapted mutant IPMDHs (Suzuki et al., 2001Go; Yasugi et al., 2001Go). Low temperature dependence of specific activity in cold-adapted mutant IPMDHs is induced by a reduction in activation enthalpy, {Delta}H!= (Suzuki et al., 2001Go; Yasugi et al., 2001Go). This tendency is also seen in natural psychrophilic enzymes (Low et al., 1973Go; Borgmann and Moon, 1975Go; Borgmann et al., 1975Go; Asegeirsson and Bjarnason, 1993; Fields and Somero, 1998Go). As shown in Figure 3Go and Table IIGo, decreased {Delta}H!= is responsible for the decrease in activation free energy, {Delta}G!= in multiple mutants, too.



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Fig. 2. Relationship between Km and kcat of mutant and wild-type IPMDHs (40°C). Closed circles, wild-type and parental mutants; open circles, multiple mutant IPMDHs. The dotted line shows the line where the ratio kcat/Km is the same as that of the wild-type IPMDH.

 


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Fig. 3. Relationship between {Delta}G!= and {Delta}H!= of mutant and wild-type IPMDHs (40°C). The dotted line shows the regression line y = 10.2x – 156.4, r2 = 0.8977.

 
Thermal stability of multiple mutants

The mutation I11V was revealed to account for the increase in thermal stability. In the case of multiple mutants that have I11V mutation, the enhanced thermal stability seemed to be inherited from I11V mutation (Table IGo). Tm was 85, 87 and 83°C in V15I, S92F and V15I+S92F, respectively, and that was increased to 87, 91 and 85°C, respectively, upon the addition of the amino acid replacement I11V (Table IGo). These results show evidence that the amino acid replacement which enhances thermal stability can be combined with other mutations to construct more thermostable mutants. Point mutation studies of triosephosphate isomerase (Williams et al., 1999Go), subtilisin (Narinx et al., 1997Go), ß-glucosidase A (Lopez-Camacho et al., 1996Go) and also IPMDH (Kotsuka et al., 1996Go; Akanuma et al., 1998Go, 1999Go; Tamakoshi et al., 2001Go) showed that these enzymes could be stabilized without losing activity. These findings suggest that the kinetic properties and thermal stability of enzymes can be modified, at least partially independently, and that different mechanisms may be involved in these two types of modification.

Thermodynamic parameters of catalytic activity of multiple mutants

Table IIIGo summarizes {Delta}{Delta}GT!=, the change in the level of free energy of the transition state induced by mutation. Multiple mutants had higher {Delta}{Delta}GT!= than the sum of {Delta}{Delta}GT!= of their parental mutants ({Delta}GI was positive). It indicates that the free energy level of the transition state was destabilized more than expected. The observation is a contrast to some examples that the free energy changes of binding of receptor and substrate show good additivity (Carter et al., 1984Go; Lowman et al., 1991Go). Wells summarized that complex additivity can be observed when mutations at sites 1 and 2 change the intramolecular interaction energy between sites (Wells, 1990Go). This can be mediated by direct steric, electrostatic, hydrogen-bonding, or hydrophobic interactions or indirectly through large structural changes in the protein, solvent shell, or electrostatic interactions. Complex additivity is most likely to occur where the sites of mutation are very close together and larger or chemically divergent side chains are introduced. If the mutation sites were accompanied by extensive structural changes in the enzyme or the Michaelis complex, the free energy change in the transition state calculated for one mutation could vary depending on the presence of the other mutations. Our results imply that the mutations interact mutually even though the sites are remote from each other, and that careful and detailed tuning of the structure is required to facilitate the higher catalytic activity.


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Table III. Additivity in free energy change of multiple mutant IPMDHs
 
Interpretation based on the structure of IPMDH

The CD spectra of I11V and multiple mutant IPMDHs were not significantly different from those of the wild-type (data not shown). They indicate that there is no significant structural difference between them, suggesting that these mutations, which caused the increase in activity at a low temperature, were not linked to large changes in the secondary structure.

The mutated residues in the mutant IPMDHs are shown in Figure 4Go. In T.thermophilus IPMDH, the adenine moiety of NAD+ has hydrophobic contact with side chains of I11, V15, L254, G255, I279 and D326 (Hurley and Dean, 1994Go). The substituted residues I11 and V15 are involved in the binding pocket of NAD+. From the crystal structural analysis, S92 is located in the {alpha}-helix from P86 to K95. This helix includes residues interacting with the nicotinamide ring of NAD+ (E87) and IPM (L90, L91, R94; Imada et al., 1991Go). In addition, three-dimensional structure analysis of the S92F mutant suggested that it could stabilize the Michaelis complex by interacting with a residue located in another domain (Hirose et al., 2001Go). V126 forms a compact hydrophobic core by constructing hydrophobic contacts with A106, V108, A123, V128, P227 and L250 (Suzuki et al., 2001Go). This hydrophobic element is succeeded by the hinge region, which contribute to the domain movement when the substrate and coenzyme bind (Hurley and Dean, 1994Go; Kadono et al., 1995Go; Imada et al., 1998Go). Because an individual effect did not simply accumulate in multiple mutants, replaced residues may interact with each other in multiple mutants directly or indirectly.



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Fig. 4. Mapping of the mutations on the tertiary structure of IPMDH monomer. Side chains of I11, V15, S92 and V126, and NAD+ are indicated by balls and sticks (PDB code 1HEX; Hurley and Dean, 1994Go). The figure was drawn using the program Insight II (Molecular Simulation Inc.).

 
Only in two of the multiple mutants, V15I+V126M and S92F+V126M, was {Delta}GI close to zero, which indicates that the effects are relatively additive. Simple additivity for {Delta}GT!= has been reported in HCA III (Tu et al., 1994Go). The additive interaction was explained to indicate that the residues which were mutated function independently to facilitate catalysis, although they need not be acting on the same step of catalytic reaction (Tu et al., 1994Go). If the mutation sites introduce no extensive structural changes in the enzyme or Michaelis complex, their effects will be independent, and the overall free energy change of the transition state in multiple mutants will be the sum of the corresponding terms for the two parental mutants. The mutations of these multiple mutants existed at spatially distant sites in different domains. V126 exists in a different domain from the domain including V15 and S92. The structural change of V126M is limited to a domain and may have little effect on the other mutations. The mutations may affect relatively independently the characteristics of the IPMDH molecule.

We have shown that it is possible to increase the catalytic activity compared with that of the wild-type at lower temperature by mutations. The way of enhancement of activity was similar to that observed in natural psychrophilic enzymes. However, it is difficult to improve the activity by combining mutations compared with parental mutants. On the other hand, the mutation which enhances thermal stability can be used to construct more thermostable mutants. It is suggested that careful tuning to optimize the structure is required to enhance the catalytic activity in contrast to thermal stability.


    Notes
 
5 To whom correspondence should be addressed. E-mail: yamagish{at}LS.toyaku.ac.jp Back


    Acknowledgments
 
This work was supported by a grant for Evolutionary Molecular Engineering from the New Energy and Industrial Technology Development Organization (NEDO) and Grants-in-Aid for Scientific Research (09558081, 10044095, 11794038) from the Ministry of Education, Science, Sports and Culture.


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Received March 9, 2001; revised May 11, 2001; accepted May 14, 2001.





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