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PEDS Advance Access originally published online on October 14, 2008
Protein Engineering Design and Selection 2008 21(12):721-727; doi:10.1093/protein/gzn054
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Random mutagenesis improves the low-temperature activity of the tetrameric 3-isopropylmalate dehydrogenase from the hyperthermophile Sulfolobus tokodaii

Michika Sasaki, Mayumi Uno, Satoshi Akanuma and Akihiko Yamagishi1

Department of Molecular Biology, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

1 To whom correspondence should be addressed. E-mail: yamagish{at}ls.toyaku.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
In general, the enzymes of thermophilic organisms are more resistant to thermal denaturation than are those of mesophilic or psychrophilic organisms. Further, as is true for their mesophilic and psychrophilic counterparts, the activities of thermophilic enzymes are smaller at temperatures that are less than the optimal temperature. In an effort to characterize the properties that would improve its activity at temperatures less than the optimal, we subjected the thermostable Sulfolobus tokodaii (S. tokodaii) 3-isopropylmalate dehydrogenase to two rounds of random mutagenesis and selected for improved low-temperature activity using an in vivo recombinant Escherichia coli system. Five dehydrogenase mutants were purified and their catalytic properties and thermostabilities characterized. The mutations favorably affect the Km values for NAD (nicotinamide adenine dinucleotide) and/or the kcat values. The results of thermal stability measurements show that, although the mutations somewhat decrease the stability of the enzyme, the mutants are still very resistant to heat. The locations and properties of the mutations found for the S. tokodaii enzyme are compared with those found for the previously isolated low-temperature adapted mutants of the homologous Thermus thermophilus enzyme. However, there are few, if any, common properties that enhance the low-temperature activities of both enzymes; therefore, there may be many ways to improve the low-temperature catalytic activity of a thermostable enzyme.

Keywords: catalytic efficiency/low-temperature activity/random mutagenesis/thermal stability/thermostable enzyme


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
An increasing number of thermostable enzymes are being isolated from thermophilic organisms. In addition to their thermostabilities, these enzymes are also unusually resistant to the effects of other protein-inactivating agents, such as organic solvents, acidic and alkaline pHs, and detergents (Suzuki et al., 2001Go). Their extreme stabilities make thermophilic enzymes attractive tools for industrial processes (Vieille et al., 1996Go; Haki and Rakshit, 2003Go; van den Burg, 2003Go). However, a crucial limitation to the design of such industrial processes is that thermostable enzymes are nearly inactive at more moderate temperatures – temperatures at which their less stable mesophilic or psychrophilic counterparts have maximum activities.

Recently, efforts have been made to improve the low-temperature catalytic activities of thermophilic enzymes. Mutations that improve the catalytic activity of Pyrococcus furiosus β-glucosidase CelB at low temperatures have been found by Lebbink et al. (2000)Go. Merz et al. (2000)Go selected Sulfolobus solfataricus (S. solfataricus) indoleglycerolphosphate isomerase mutants that are catalytically more active at 37°C than is the wild-type enzyme. Low-temperature adaptation of the thermophilic Thermus thermophilus (T. thermophilus) xylose isomerase’s enzymatic activity has also been reported (Lönn et al., 2002Go). Random mutagenesis of a rationally designed, low-temperature adapted mutant of the extremely thermostable Thermotoga neapolitana xylose isomerase produced additional mutants with improved low-temperature adaptations (Sriprapundh et al., 2003Go). While these examples clearly demonstrate that one or a small number of mutations can improve low-temperature activity, predictive rules, based on physical and/or chemical guidelines, have not yet been established.

3-Isopropylmalate dehydrogenase (IPMDH, EC 1.1.1.85 [EC] ), the product of the leuB gene, is an enzyme involved in leucine biosynthesis. The leuB gene has been cloned from a variety of micro-organisms including the extreme thermophile, T. thermophilus HB8 (Tanaka et al., 1981Go) and the hyperthermophile Sulfolobus tokodaii (S. tokodaii) (Suzuki et al., 1997Go). The catalytic properties, the thermal stability, and the tertiary structure of the unusually thermostable T. thermophilus IPMDH are well characterized (Yamada et al., 1990Go; Imada et al., 1991Go). Some T. thermophilus IPMDH mutants that are catalytically more active than the wild-type enzyme at temperatures ranging from 30 to 40°C have been isolated from a library composed of randomly mutated leuB genes (Suzuki et al., 2001Go; Yasugi et al., 2001Go). Detailed analyses of the kinetic properties of these low-temperature adapted mutants show that the effects caused by their mutations are of two types. Some substitutions contribute to an increased kcat value; whereas, others only result in an improved Km value for the coenzyme NAD (nicotinamide adenine dinucleotide). To date, no T. thermophilus IPMDH mutant exists that has both improved kcat and Km values.

For the study reported herein, we focused on the most thermostable IPMDH characterized to date, which is that of S. tokodaii. Its biophysical properties and tertiary structure are known (Suzuki et al., 1997Go). The thermostability of this enzyme is greater than that of the T. thermophilus enzyme by 10°C. The enzyme is composed of four identical subunits of 337 amino acid residues (PDB ID: 1WPW). Conversely, other IPMDHs with known three-dimensional structures, including the T. thermophilus enzyme, are homo-dimeric proteins. Herein we report a random mutagenesis of the S. tokodaii leuB gene that was followed by selection and purification of mutants with improved low-temperature catalytic activities. The positions and physical characteristics of the mutations are compared with those that contribute to the low-temperature adaptation of T. thermophilus IPMDH.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Random mutagenesis by error-prone PCR

The S. tokodaii leuB gene was cloned into the NdeI-EcoRI site of the pET21c (+) plasmid. The resulting plasmid is named pE7-SB21. To randomly mutate and amplify the leuB gene, error-prone polymerase chain reaction (PCR) mutagenesis was performed using a slightly modified version of the method of Leung et al. (1989)Go, the pE7-SB21 plasmid, and the PCR primers T7P (5'-TAA TAC GAC TCA CTA TAG GG-3') and T7T (5'-CTA GTT ATT GCT CAG CGG T-3'). Gene Taq DNA polymerase (Nippon gene, Tokyo) was used, as was MnCl2 of 0.2 mM. Gene amplification involved an initial 95°C incubation of the PCR mixture for 1 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 1 min, and polymerization at 72°C for 5 min. Finally, the mixture was incubated at 72°C for 20 min. The randomly mutated leuB genes were digested with XbaI and EcoRI and then ligated into pET21c (+). The ligates were amplified in Escherichia coli (E. coli) JM109. The recovered plasmids define the randomly mutated leuB gene library (hereafter referred to as ‘gene library #1’).

Selection

The leuB-deficient E. coli strain MA153, which was constructed from E. coli BL21 (DE3) by deleting its chromosomal leuB gene, was transformed with gene library #1. The transformed cells were plated onto agar containing M9 minimal medium supplemented with 4 µg/ml isopropyl-thio-β-D-galactopyranoside (IPTG) and 50 µg/ml ampicillin. After 3 days of culture at 25°C, colonies that contained a plasmid from gene library #1 and grew faster than did the MA153 strain containing the S. tokodaii wild-type gene, were selected for further study.

Growth measurements

Plasmid DNAs were recovered from the selected transformed E. coli MA153 colonies. E. coli MA153 cultures were then transformed with the isolated plasmid DNAs, plated onto agar containing M9 minimal medium, supplemented with 50 µg/ml ampicillin, and incubated at 37°C for 2 days. Individual colonies were picked and used to inoculate liquid M9 minimal medium supplemented with 50 µg/ml ampicillin and 4 µg/ml IPTG. These test-tube cultures were shaken at 25°C. Inocula of E. coli MA153 harboring the wild-type leuB gene was also cultured in liquid M9 minimal medium in the absence or in the presence of 100 µg/ml leucine, under otherwise identical conditions as described for the selected colonies. During the incubations, which lasted 54 h, culture samples were removed and each sample was assayed for cell growth density by measuring its absorbance at 600 nm.

Expression levels of S. tokodaii IPMDH and its mutants

E. coli MA153 strains transformed with the pET21c (+) plasmid carrying the S. tokodaii leuB gene or a mutated gene were cultured at 25°C in liquid M9 minimal medium supplemented with 50 µg/ml ampicillin and 4 µg/ml IPTG. When a culture reached an OD600 of 0.2, its cells were harvested by centrifugation and disrupted by sonication. The same quantities of proteins in the cell lysates were separated by 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and protein bands were stained with Coomassie Brilliant Blue R-250.

Second round of mutagenesis and selection

The leuB genes encoding for the IPMDH mutants (named for their mutations: F3S, R284G and L288P) were subjected to a second round of error-prone PCR and genetic selection. The PCR conditions were the same as those of the first round of error-prone PCR as described earlier. For selection, the leuB-deficient E. coli MA153 was transformed with the second-round randomly mutated leuB gene library (hereafter called gene library #2) and then plated onto agar containing M9 minimal medium supplemented only with 50 µg/ml ampicillin. The plates were incubated at 25°C and colonies that grew faster than the MA153 strain harboring the S. tokodaii wild-type leuB gene were selected after 3 days of culture.

IPMDH purification

To prepare and purify each of the S. tokodaii wild-type and mutant IPMDHs, an E. coli BL21-CodonPlus (DE3)-RIL inoculum (Stratagene, La Jolla, CA, USA) harboring the respective expression plasmid was cultivated in 2YT medium supplemented with ampicillin (50 µg/ml). Overexpression was induced by adding IPTG to a final concentration of 1 mM when the culture reached an OD600 of 0.6. After an additional 3 h at 37°C, cells were harvested and disrupted by sonication. The soluble fractions were each isolated after centrifugation at 60 000g for 20 min. The supernatants were individually heated at 75°C for 20 min and centrifuged at 60 000g for 20 min. To purify each enzyme, its respective supernatant was successively chromatographed over DEAE cellulose (DE52, Whatman, Tokyo) and butyl-Toyopearl (Tosoh, Tokyo). The proteins were all considered to be homogeneous based on the results of SDS–PAGE.

Analytical gel filtration

For analytical gel filtration, protein, in an initial volume of 0.2 ml, was eluted at a flow rate of 0.5 ml/min over Superdex 200 (1.0 x 30 cm column dimensions; GE Healthcare Biosciences, Piscataway) that was equilibrated with 20 mM potassium phosphate (pH 7.6) and 300 mM NaCl. Apparent molecular masses were determined using the protein elution volumes and a calibration curve was produced using proteins of known molecular masses and elution volumes.

Enzyme assays

Enzymatic activities were determined using the results of an assay where the increase in absorbance at 340 nm was measured; this absorbance increase corresponds to the production of reduced NAD (NADH), one of the products of IPMDH catalysis (Yamada et al., 1990Go). The assay buffer was 50 mM HEPES (pH 7.5), 100 mM KCl, 5 mM MgCl2, 0.1 mM D-3-isopropylmalate (D-3-IPM; a substrate of IPMDH), and 5.0 mM NAD. One enzyme unit equals 1 µmol NADH formed per minute (Yamada et al., 1990Go).

The values of the Michaelis constant (Km) for the substrate D-3-IPM and the catalytic constant (kcat), were determined using the results of steady-state experiments with assay buffers of 50 mM HEPES (pH 7.5), 100 mM KCl, 5 mM MgCl2, 5 mM NAD, and D-3-IPM concentrations between 2 and 30 µM. To determine the Km value for NAD, the coenzyme concentration was varied between 60 and 2500 µM, while the D-3-IPM concentration was fixed at 0.1 mM. The kinetic constants were obtained by nonlinear least-square fitting of the steady-state velocity data to the Michaelis–Menten equation using the Enzyme Kinetics module of SigmaPlot (Systat Software, Richmond, VA, USA).

Thermal stability measurements

Thermal denaturations were monitored using the changes in the ellipticity of the proteins at 222 nm, which were recorded with a J720C spectropolarimeter (JASCO, Hachioji). Each enzyme (0.25 mg/ml) was dissolved in 20 mM potassium phosphate (pH 7.0), 0.5 mM ethylenediaminetetraacetic acid. The temperature of an enzyme solution was controlled using a thermostated circulating water bath and a programmable temperature controller. The precise temperature was monitored with the probe of a thermocouple inserted directly into the enzyme solution. The scan rate was 0.5°C/min.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Construction of a S. tokodaii leuB gene library and selection for IPMDH activity at 25°C

The maximum activity of S. tokodaii IPMDH occurs at 95°C and it retains only 2% of that activity at 25°C (Suzuki et al., 1997Go). To improve the catalytic efficiency of this hyperthemophlic enzyme at lower temperatures, error-prone PCR and genetic selection were used to find mutated S. tokodaii IPMDHs that are more active at lower temperatures. First, the S. tokodaii leuB gene was amplified with Taq DNA polymerase and PCR conditions that promoted error-prone polymerization. To select for mutated leuB genes that encode IPMDHs with enhanced catalytic activities at 25°C, the leuB-deficient E. coli strain MA153 was first transformed with gene library #1, which theoretically contains 1.9 x 105 different variants, and then plated onto agar containing M9 minimal medium. Fourteen colonies that grew faster than those carrying the plasmid containing the S. tokodaii wild-type leuB gene were selected after 3 days at 25°C. These faster-growing colonies were assumed to contain mutated leuB genes that encode for enzymes with improved activities at 25°C. The plasmid DNAs were isolated from these colonies and their leuB genes sequenced. The sequences of the original and mutated codons, and the types and positions of their corresponding mutated residues are listed in Table I.


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  		Table I. Low-temperature adapted S. tokodaii IPMDH mutants produced using random mutagenesis and selection

 
Three of the mutants, named according to their residue substitutions (F3S, R284G and L288P), occurred several times and therefore were chosen for additional study. The three E. coli MA153 strains each containing one of the single-site mutated leuB genes grew more rapidly than did E. coli MA153 containing the wild-type leuB gene in leucine-free M9 minimal liquid medium (Fig. 1). A mutation could cause an increased growth rate by improving the catalytic constants, increasing the expression efficiency, or increasing the plasmid copy number. To eliminate the latter two possibilities, we compared the levels of the expressed wild-type and mutated proteins contained in equal amounts of whole-cell protein extracts. As seen in Fig. 2, the amounts of wild-type and mutant IPMDHs seem to be similar, suggesting that the increased growth rates of the E. coli MA153 strains containing the mutant IPMDHs are not due to increased expression levels of the mutant enzymes.


Figure 1
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  		Fig. 1. Growth curves at 25°C for the leuB-deficient E. coli strain MA153 transformed with plasmids containing the genes for S. tokodaii IPMDH or its mutants. Growth of MA153 containing the gene for the wild-type enzyme was measured in the presence and absence of 100 µg/ml leucine in M9 minimal medium. Growth of the other MA153 strains was measured without supplemented leucine in M9 minimal medium. Growth rates were measured by recording the cultures’ absorbances at 600 nm (A600) at the indicated times. Results are the means of at least five independent measurements.

 

Figure 2
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  		Fig. 2. An SDS–PAGE after separation of the proteins of the cell lysates of the E. coli strain MA153 transformed with plasmids containing the genes for S. tokodaii IPMDH or its mutants. The arrow indicates the position expected for IPMDH (37 kDa). Lane M, molecular mass standards, bands from top to bottom correspond to 100, 70, 50, 40, 30, 25 and 20 kDa; lane 1, wild-type; lane 2, F3S; lane 3, R284G; lane 4, L288P; lane 5, F3S + S232G; lane 6, F3S + L288P.

 
Second-round random mutagenesis and selection

The three leuB genes, containing a F3S, R284G or L288P mutation, were subjected to a second round of error-prone PCR and genetic selection. For this experiment, E. coli MA153 was transformed with gene library #2, which theoretically contains 1.0 x 105 different leuB mutants, and was grown at 25°C on agar containing M9 minimal medium. Two fast-growing colonies were selected after 3 days of culture at 25°C. A leuB gene isolated from one colony contains the F3S mutation and the novel mutation, S232G (Table I). The leuB gene of the other colony contains the F3S and L288P mutations. Both of these MA153 strains each containing a doubly mutated leuB gene grew more rapidly than did E. coli MA153 containing the wild-type leuB gene in leucine-free M9 minimal liquid medium (Fig. 1).

Oligomeric structure of the low-temperature adapted variants

The three S. tokodaii IPMDH mutants with the single amino acid substitutions (F3S, R284G and L288P) and the two double-mutation variants (F3S + L288P and F3S + S233G) were expressed in E. coli, purified, and characterized. The oligomeric states of the S. tokodaii wild-type enzyme and the mutants were investigated using analytical gel filtration. The elution profiles show that each IPMDH migrates as a single elution peak with a retention volume corresponding to the molecular weight expected for tetrameric IPMDH (data not shown). Thus, the mutations do not affect the oligomeric states of the enzymes.

Temperature dependence of the specific activities

Figure 3A depicts the temperature dependencies of the wild-type and mutant IPMDHs’ specific activities. The temperature dependencies are similar for all IPMDHs in that the specific activities increase as the temperature increases >30°C. The specific activities of the mutant enzymes, excluding F3S + S232G, are 1.15–1.46-fold greater than that of the wild-type enzyme at 40°C. Because the L288P and F3S + L288P mutants also have enhanced specific activities at very high temperatures, it is possible to improve low-temperature activity without sacrificing high-temperature activity. Activation energies (Ea) for the specific activity were calculated from the slopes of the Arrhenius plots for the temperature range of 30–70°C (Fig. 3B). Whereas the F3S, L288P and F3S + L288P mutants show only marginally or slightly reduced Ea values (65.1, 62.7 and 62.6 kJ/mol, respectively) compared with that of the wild-type enzyme (66.4 kJ/mol), the R284G and F3S + S232G mutants show significantly reduced Ea values (58.6 and 53.3 kJ/mol, respectively). The smaller Ea value of F3S + S232G may be at least partially responsible for the significantly improved growth characteristic of the F3S + S232G-containing MA153 strain (Fig. 1).


Figure 3
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  		Fig. 3. (A) A plot of the specific activities of the wild-type and mutant S. tokodaii IPMDHs. The specific activity assay was performed using a mixture of 0.1 mM D-3-IPM, 5 mM NAD, and 7 µM (30–50°C) or 1.5 µM (60–90°C) protein. All values are the averages of three determinations. The specific activities of all the mutants, excepting F3S + S232G, are greater than that of the wild-type enzyme at 40°C (inset): wild-type, 0.99 ± 0.02 units/mg; F3S, 1.14 ± 0.01 units/mg; R284G, 1.34 ± 0.02 units/mg; L288P, 1.45 ± 0.04 units/mg; F3S + S232G, 0.93 ± 0.01 units/mg; F3S + L288P, 1.37 ± 0.06 units/mg. (B) Arrhenius plots for the specific activities of wild-type and mutant IPMDHs. To avoid the effect of protein denaturation, the specific activities at 80 and 90°C were omitted. Activation energies calculated from the slopes of the plots are as follows: wild-type, 66.4 ± 2.3 kJ/mol; F3S, 65.1 ± 1.7 kJ/mol; R284G, 58.6 ± 1.7 kJ/mol; L288P, 62.7 ± 2.4 kJ/mol; F3S + S232G, 53.3 ± 2.7 kJ/mol; F3S + L288P, 62.6 ± 1.5 kJ/mol. The color code is: black, wild-type; red, F3S; blue, R284G; green, L288P; purple, F3S + S232G; magenta, F3S + L288P.

 
Kinetic parameters

The kinetic parameters of the IPMDHs were calculated using steady-state experimental data obtained at 25, 40 and 70°C (Table II). The single-mutation IPMDH, L288P and the double-mutation enzyme, F3S + S232G, have improved Km values for 3-IPM at 40°C. Moreover, significantly improved Km values for NAD at 25°C are observed for all of the mutated IPMDHs, except for F3S + L288P, which has only a marginally improved Km value for NAD. The double-mutation enzyme, F3S + S232G, has the most unfavorable kcat values at all temperatures, but also the most improved Km's for both 3-IPM and NAD. The single-mutation variants, R284G and L288P, and the double-mutation variant, F3S + L288P, have slightly increased kcat values at 25 and 40°C in comparison with those of the wild-type enzyme. Thus the mutated IPMDHs adapt to low temperature by having improved coenzyme affinities and/or at least slightly increased turnover numbers. All of the mutated IPMDHs also show improved Formula values at the low temperatures. The most improved Formula values are those found for the F3S mutant at 25°C and for the F3S + S232G mutant at 40°C – 1.8 and 2.0-fold increases, respectively.


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  		Table II. Kinetic constants for the wild-type and mutant S. tokodaii IPMDHs

 
Thermal stability

To determine if the mutant IPMDHs are as resistant to thermal denaturation as the wild-type is, temperature-induced unfolding of the S. tokodaii enzymes, as well as that of wild-type T. thermophilus IPMDH, were monitored using CD spectroscopy at 222 nm, since the ellipticity at 222 nm is a measure of protein secondary structure content. To compare the relative stabilities of the S. tokodaii IPMDHs, their Tm values—Tm is the temperature at which 50% of a protein population is unfolded—were determined. All of the mutants are less stable than the wild-type enzyme is and have decreased Tm values of 4.4–10.2°C (Fig. 4). Nevertheless, the mutants are still extremely thermostable enzymes since their unfolding curves are similar to that of T. thermophilus IPMDH.


Figure 4
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  		Fig. 4. Thermal melting profiles for the wild-type and mutant S. tokodaii IPMDHs. Ellipticities were monitored at 222 nm. The scan rate was 0.5°C/min. The solutions were 0.25 mg/ml protein, 20 mM potassium phosphate (pH 7.0), 0.5 mM ethylenediaminetetraacetic acid. The thermal unfolding curve for T. thermophilus IPMDH is also shown in gray for comparison purposes. Symbols: closed circles, S. tokodaii IPMDH; open triangles, F3S; open diamonds, R284G; open squares, L288P; closed triangles, F3S + S232G; closed squares, F3S + L288P; open circles, T. thermophilus IPMDH.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Evolutionary molecular-engineering is a powerful approach that produces proteins with modified properties. It mimics the natural evolution process by using a combination of (random) mutagenesis and selection (or screening) of nucleic acids or proteins. A big advantage of this approach is that the modified properties arise without a detailed knowledge of the structure and/or function of the protein under study (reviewed by Höcker (2005Go) and references therein). Our results clearly demonstrate that evolutionary molecular-engineering mimics the presumed evolutionary pressures experienced by proteins in nature. Using an evolutionary molecular-engineering technique, we have been able to improve the catalytic efficiency of S. tokodaii IPMDH at low temperatures; similar results have been reported for other enzymes (Taguchi et al., 1998Go; Lebbink et al., 2000Go; Merz et al., 2000Go; Wintrode et al., 2000Go; Roovers et al., 2001Go; Lönn et al., 2002Go; Sriprapundh et al., 2003Go; Prieto et al., 2008Go).

We mapped the mutations found for the low-temperature adapted S. tokodaii and T. thermophilus IPMDHs (Suzuki et al., 2001Go; Yasugi et al., 2001Go) onto their crystal structures (Fig. 5). A defining structural feature of the S. tokodaii enzyme is its tetrameric quaternary structure, which is not present in less thermostable IPMDHs, including the T.thermophilus enzyme. For Pyrococcus furiosus β-glucosidase (Lebbink et al., 2000Go), mutations that cause a decrease in the optimal temperature for activity are found at the subunit interface, suggesting that interface contacts might modulate temperature adaptation. However, although the IPMDH mutations reported herein are distributed throughout the enzyme, they are not found at the interface. Therefore, for S. tokodaii IPMDH, altering the subunit contacts seems not to be a means of improving S. tokodaii IPMDH low-temperature activity.


Figure 5
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  		Fig. 5. A structural map of the positions of the S. tokodaii and T. thermophilus IPMDH mutations. S. tokodaii IPMDH is a homo-tetramer (left) and T. thermophilus IPMDH is a homo-dimer (right). The subunits of the enzymes are each colored differently. In one subunit of each enzyme the positions of the mutations are labeled. The residues that interact with 3-IPM and NAD are colored blue and red, respectively. The figure was drawn using PyMOL (http://www.pymol.org).

 
Our kinetic analyses show that the increases in the catalytic activities of the mutants are partly caused by smaller Km values for NAD. Conversely, some of the IPMDH mutations unfavorably affect the 3-IPM Km's. The 3-IPM and NAD binding pockets are spatially separated on the enzyme’s surface (Hurley and Dean, 1994Go; Kadono et al., 1995Go; Imada et al., 1998Go). Therefore, it is not surprising that changes in the Km's for the substrate and the coenzyme are not coupled. Our results also suggest that favorable binding of substrate to enzyme is not required for low-temperature adaptation. The same observation holds for the low-temperature adapted T. thermophilus IPMDH mutants (Suzuki et al., 2001Go; Yasugi et al., 2001Go). It should be noted that NAD is a cofactor used in many different biological reactions. The concentration of NAD in E. coli should therefore be independent of the level of IPMDH activity. Conversely, the concentration of the specific substrate, 3-IPM, should be dependent. When IPMDH activity is low, 3-IPM should accumulate. Consequently, as proposed by Suzuki et al. (2001)Go, low-temperature adaptation should be modulated by the affinity of the enzyme for NAD and not that of 3-IPM.

Many authors have suggested that the degree of an enzyme’s conformational flexibility correlates directly with its rate of catalysis (Gerday et al., 1997Go; Fields and Somero, 1998Go; Cavicchioli et al., 2002Go; Georlette et al., 2003Go). Thermophilic enzymes are intrinsically rigid at low temperatures; it follows therefore that elevated temperatures provide the necessary energy to increase structural flexibility (Jaenicke, 2000Go). NAD binding to T. thermophilus IPMDH induces a large structural change in a loop (Hurley and Dean, 1994Go; Imada et al., 1998Go). A similar structural change is expected for the corresponding loop of S. tokodaii IPMDH upon NAD binding. Perhaps, therefore, mutants that exhibit an increased turnover rate at 25°C, also exhibit an increased flexibility in the aforementioned loop, which would provide the decrease in transition-state energy needed for enhanced catalysis.

Since the crystal and/or solution structures of the mutants are not known, the relationships between structure and activity cannot be explored at the molecular level. Instead, using the crystal structures of the wild-type IPMDHs as guides, we tabulated the changes in accessible surface areas and side-chain volumes, and the distances from the active sites for the S. tokodaii and T. thermophilus mutants (Table III). For S. tokodaii IPMDH, residues 3, 284 and 288 are far away from the active site and are solvent exposed. Conversely, residue 232 is near the active site and is buried. Similarly, the mutated residues of T. thermophilus IPMDH are both solvent exposed and buried; and both near to and far from the active site. While all of the S. tokodaii mutated residues have side chains of smaller volumes than those that they replace, the same cannot be said for the T. thermophilus mutations. Therefore, we cannot at this time suggest a general rule(s) to guide the design of low-temperature adapted mutants of thermostable enzymes. Instead, our results and those of others (Lönn et al., 2002Go; Coker and Brenchley, 2006Go) may be interpreted to suggest that low-temperature adaptations are the consequences of many different types of subtle conformational changes.


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  		Table III. Physical parameters for the mutated residues of the S. tokodaii and T. thermophilus mutant IPMDHs

 
All of the S. tokodaii mutants are less resistant to temperature denaturation than is the wild-type IPMDH (Fig. 4). However this correlation cannot be extended to other thermostable enzymes since most of the low-temperature adapted T.thermophilus mutants are as stable as the wild-type IPMDH is (Suzuki et al., 2001Go; Yasugi et al., 2001Go) and as are the low-temperature adapted mutants of P. furiosus β-glucosidase (Lebbink et al., 2000Go), S. solfataricus indoleglycerolphosphate synthase (Merz et al., 2000Go), and subtilisin (Kano et al., 1997Go). While it might be concluded from the results of the aforementioned studies on enzymes other than S. tokodaii IPMDH that enzymatic activity is independent of conformational stability, the same cannot be said for S. tokodaii IPMDH. Protein structures suit their physiological environments; therefore, the S. tokodaii IPMDH structure must have been evolutionarily optimized for high temperatures. Because the S. tokodaii mutants of this study were not selected for their resistance to thermal denaturation, it is not surprising that they are less thermostable. Nevertheless, their thermostabilities are similar to that of the thermophilic T. thermophilus IPMDH. Accordingly, as suggested before (Sriprapundh et al., 2003Go), it is apparent that the catalytic activity of a thermostable enzyme at lower temperatures can be improved without considerable loss of its extreme stability.


   Footnotes
 
Edited by JacQues Fastrez


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received June 2, 2008; revised September 8, 2008; accepted September 15, 2008.


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