1 Department of Life Science, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8501, 2 Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Horinouchi 14321, Hachioji, Tokyo 192-0392 and 4 Institute of Applied Biochemistry, University of Tsukuba, Tennoudai 111, Tsukuba 305-8572, Japan
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Abstract |
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Keywords: cold adaptation/3-isopropylmalate dehydrogenase/mutagenesis/thermophile enzyme
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Introduction |
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We have recently reported that the mutant enzymes that show enhanced activity at low temperatures were isolated from a random mutant library of IPMDH of an extreme thermophile, Thermus thermophilus (Suzuki et al., 2001; Yasugi et al., 2001
). One of the mutants, Val126Met, showed a 7.5-fold-improved kcat value with an increased Km value for the coenzyme NAD at 40°C (Suzuki et al., 2001
). Thermodynamic analysis revealed that the cold adaptation of this mutant enzyme is due to a decrease in the free energy change for coenzyme binding and that it is enthalpy driven. The kinetic characteristics (lower
G
and
H
) were similar to those reported for some naturally occurring cold-adapted enzymes (Lonhienne et al., 2000
). Although some strategies for low-temperature adaptation, such as optimizing electrostatic interactions (Brandsdal et al., 2001
), increased molecular flexibility (Lonhienne et al., 2000
) and reduced number of interactions between structural domains or subunits (Smalas et al., 2000
), have been proposed, it is difficult to predict the contribution of each amino acid residue, because of significant differences between the primary structures of the enzymes investigated. The Val126Met mutant is expected to provide insights into understanding the adaptation strategy, because it possesses only one amino acid substitution.
The Val126 residue is located in the hinge region far from the binding pockets for IPM or NAD and its side chain is buried in a hydrophobic core (Imada et al., 1991; Hurley and Dean, 1994
). Structural determination of the mutant enzyme revealed that the substitution causes structural changes in a loop structure of the hinge and consequently prevents the loop from forming the closed configuration of the NAD-binding pocket (Hirose et al., 2001
). In the present study, a number of mutant IPMDHs, Val126Gly, Ala, Ser, Thr, Glu, Ile, Leu and Phe, were prepared by site-directed mutagenesis. The molecular mechanism of enzymatic adaptation to cold environments is discussed on the basis of analyses of the mutant IPMDHs.
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Materials and methods |
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DNA manipulations were performed following the method reported in the literature (Sambrook et al., 1989). Site-directed mutagenesis of the T.thermophilus leuB gene was performed by the method of Kunkel (Kunkel and Roberts, 1987), using plasmid pNV119 (Tamakoshi et al., 1995
) as a template with a synthetic oligonucleotide, 5'-CGATGAGGACGTCNNNCCCGCGGGCGATCTCC-3'. The NNN region is designed as follows: Gly, CCC; Ala, TGC; Ser, AGA; Thr, CCG; Glu, TTC; Leu, CTG; Ile, ATC; Phe, TTG. The nucleotide sequences of the resulting clones were confirmed by sequencing.
The wild-type and resulting mutant IPMDHs were overexpressed in a leuB-deficient Escherichia coli strain, OM17 (Tamakoshi et al., 1995) and purified to homogeneity, as performed previously (Yamada et al., 1990
).
Analytical procedures
Specific activity was measured in 50 mM HEPESKOH buffer (pH 8.0) containing 100 mM KCl, 5.0 mM MgCl2, 5.0 mM NAD and 1.0 mM D,L-3-isopropylmalate (IPM), as described previously (Suzuki et al., 2001). The initial rate of the reaction was measured by monitoring spectrophotometrically the reduction of NAD at 340 nm. Kinetic parameters, kcat and Km for NAD, were determined by measuring the initial rates of the catalytic reaction with a saturated concentration of IPM (1 mM) and variable concentration of NAD. Km values for IPM were determined in the presence of 5 mM NAD. Thermodynamic parameters of activation of the reaction,
G
and
H
, were derived, according to the relationships presented in the literature (Lehrer and Barker, 1970
), from the apparent Vmax values determined between 20 and 95°C in the presence of 1 mM IPM and 5 mM NAD.
Heat-induced melting profiles of the secondary structure were obtained by measuring the circular dichroism at 222 nm at a scanning rate of 1°/min in a Model 720 spectropolarimeter (JASCO, Japan) using a cuvette with a 1 mm pathlength, as performed previously (Hayashi-Iwasaki et al., 1996). Temperature was monitored using a CuNi thermocouple inserted in the cuvette.
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Results and discussion |
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As shown in Figure 1A, the Val126 residue is located in domain II (Imada et al., 1991
; Hurley and Dean, 1994
). The residue is far from the binding site for NAD: the distance between the C
atom of Val126 and the C4 atom of the nicotinamide ring is 27 Å and between the C
atom of Val126 and the N3 atom of the adenine ring is 23 Å. The residue is also buried in the interior of the enzyme molecule and forms a hydrophobic packing with the surrounding hydrophobic residues, Ala106, Val108, Ala123, Val128, Pro227 and Leu250 (Figure 1B
). Therefore, the valine residue does not directly interact with either IPM or NAD, even when the enzyme forms a closed conformation (Imada et al., 1998
).
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Effect of the mutations on thermal stability of IPMDH
Temperature-induced melting profiles of the secondary structure were analyzed by monitoring changes in circular dichroism (CD) at 222 nm, as performed previously (Hayashi-Iwasaki et al., 1996). The mutant IPMDHs underwent a temperature-induced cooperative transition from the native state to the unfolded state, as observed for the wild-type enzyme. Half-denaturation temperatures (T
) of the mutant IPMDHs at Val126 were as follows: wild-type (Val), 87; Gly, 82; Ala, 85; Ser, 74; Glu, 77; Met, 87; Leu, 82; Ile, 82; and Phe, 80°C. The T
values are plotted against the van der Waals volume of residue 126 in Figure 2
. Among these mutant enzymes, two with hydrophilic residues, Val126Ser and Val126Glu, lost significant thermal stability. The loss of stability can be explained by the general rule that the introduction of a polar or a charged residue into a hydrophobic core induces destabilization of the structure (Lim et al., 1992
).
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Catalytic activity modulated by the mutation
The effect of the substitution of Val126 on the catalytic function was investigated by determining the steady-state kinetic parameters (Table I). Mutant IPMDHs showed lower or higher kcat values than the wild-type depending on the mutations at 60°C. The Km values for IPM and NAD were also changed by the mutations. Mutants that showed higher kcat values tended to show higher Km values. Changes in Km values for IPM and NAD were similar in respective mutants, suggesting a similar effect of the mutation on the binding pockets for both IPM and NAD. These results indicate that the structural perturbation around residue 126 is indirectly propagated to the binding residues with IPM and NAD. The effect of the mutation Val126Met was different from those of other mutants and significantly higher Km for NAD was obtained for this mutant.
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Similar tendencies were seen for kcat and Km for NAD at 40°C. The degree of improvement in kcat was higher than that at 60°C. In the analysis, Km for IPM was too low to be estimated accurately at 40°C and therefore was not determined.
Profound aspects could be seen when the kinetic parameters were plotted against the van der Waals volume of residue 126 (Figure 3). The parameters showed a good correlation with the volume: the larger the volume, the greater were the kinetic parameters. In contrast to the thermal stability of these mutants (Figure 2
), no obvious optimal volume was found in these plots for the residues tested. Moreover, two mutants with a polar residue at position 126 (Val126Ser and Val126Glu) did not exhibit any deviation from the dependence. The results indicate that the changes in the kinetic parameters are affected by a volume-based molecular mechanism. The kinetic parameters were also slightly affected by the shape of the side chain; the parameters were different between Val126Leu, Val126Ile and Val126Met. However, the packing volume is the dominant factor controlling the kinetic parameters. The effect of the volume on the catalytic function has been reported for several proteins (Imoto et al. 1994
; Han et al., 1997
; Uchida et al., 1997
; Cantu and Palzkill, 1998
; Johansson et al., 1998
). Although the kinetic parameters are reported for some of those enzymes, thermodynamic parameters have not yet been analyzed.
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Thermodynamic parameters of activation of the catalytic reaction in mutant IPMDHs were determined. The activation enthalpy (H
) of the reaction was affected by the mutation, which also correlated with the van der Waals volume of residue 126 regardless of the nature of the side chain (Figure 4A
). The changes in the activation Gibbs free energy (
G
) were plotted against activation enthalpy (
H
) (Figure 4B and C
). Except for Val126Met, the
G
values of the mutant IPMDHs showed linear correlations to
H
, indicating that the decreases in
G
were mainly achieved by the decrease in
H
.
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Figures 3C and 4 also show that the degree of cold adaptation of IPMDH appears to be related to the residue volume at position 126. The relative improvement of kcat at 40°C was higher in the mutants with larger residues at position 126 (Figure 3C
). The activation free energy (Figures 4B and C
) and enthalpy (Figure 4A
) are lower in the mutants with larger residues at position 126. Borgmann et al. hypothesized that the decrease in
H
values can be ascribed to the decreasing number of weak interactions in the enzymesubstrate complex (Borgmann and Moon, 1975
; Borgmann et al., 1975
; Smalas et al., 2000
). Higher kcat values found in some mutants (Val126Met, Leu, Ile and Phe) may be ascribed to the decreased enzymeligand interaction, i.e. the destabilized ternary complex represented by higher Km values.
Val126Met mutant deviated significantly from the volume dependence of H
(Figure 4A
). The decrease in
H
is compensated for by the decrease in
S
, resulting in a smaller decrease in
G
than that expected from the decrease in
H
(Figure 4B and C
). Km for NAD in Val126Met mutant is especially higher than the other mutants (Figure 3
). These results suggest that the ternary complex in Val126Met mutant is much more destabilized than expected from its residue volume. Although the reason for destabilization of ternary complex is not clear, it may be ascribed to the shape of the Met residue.
Molecular basis of the changes in the catalytic function
NAD binding to T.thermophilus IPMDH has been known to induce a large structural change in a loop structure (Leu250Leu254, see Figure 1B) (Hurley and Dean, 1994
; Imada et al., 1998
). A similar structural change in the loop was also observed when IPM was bound to the enzyme (Kadono et al., 1995
; Imada et al., 1998
). This loop forms a hinge structure connecting two domains of this enzyme and plays an important role in the formation of the open/closed configurations of the domains during catalysis (Kadono et al., 1995
). A recent X-ray crystallographic study revealed that this hinge loop undergoes a structural perturbation upon substitution of residue 126, because the side chain of residue 126 is in van der Waals contact with the side chain of Leu250 in the hinge loop (Figure 1B
) (Hirose et al., 2001
). Volume-dependent changes of the kinetic and thermodynamic parameters found in Val126 mutants are indicative of the structural perturbation in the hinge. Therefore, changes in the catalytic trait found in the present study can be ascribed to the alteration of the hinge motion and/or induced fit to the substrate and the coenzyme. Close correlation between the hinge motion and the catalytic function has also been observed in some other enzymes, such as dihydrofolate reductase (Tan and Freisheim, 1990; Ahrweiler and Frieden, 1991
), ornithine transcarbamoylase (Ha et al., 1997
) and F1-ATPases (Abrahams et al., 1994
; Masaike et al., 2000
). Kinetic and thermodynamic consequences of the mutant IPMDHs further support the structural change. First, the mutation at position 126 induced the changes in Km values for IPM and NAD, to similar extents, except for the Met mutant. Two binding pockets for IPM and NAD are separately located on the enzyme surface (Figure 1A
) and, therefore, equal changes must be achieved by altering the structure that is not directly related to the binding of one of these ligands. Second, the kinetic and thermodynamic analyses suggested that changes occurred in the stability of the ternary complex. These results suggest that the structural change in the hinge caused by overcrowding the hydrophobic core is an adaptive strategy of this enzyme to low temperatures.
In the catalytic reaction of T.thermophilus IPMDH, the rate-limiting step has been suggested to be product release (Dean and Dvorak, 1995; Suzuki et al., 2001
) and, therefore, the kcat value is determined by the rate of product release. Hence the changes in the kcat values, together with those in the Km values, can be attributed to the alteration of the hinge motion, because the structural movement between open/closed conformations appears to be involved in ligand binding/release (Hurley and Dean, 1994
; Kadono et al., 1995
; Imada et al., 1998
). A tentative explanation might be that the high catalytic activity with high Km values, as found in mutants with high volumes, may be a consequence of the overcrowding of the hydrophobic core by the large side chain, allowing the open configuration of the hinge and product release to occur more readily at the cost of a decrease in the efficiency of capturing IPM and NAD.
The mode of the effect of residue volume may depend on the characteristics of the reaction: rate-limiting step, the characteristics of the substrate and the relative geometry between the active site and the mutation site. In human glutathione transferase P1-1, a change in the side chain of residue 105 seemed primarily to cause changes in the Km value, whereas the kcat value was not distinctively affected when 1-chloro-2,4-dinitrobenzene was used as substrate. With other substrates, both the kcat and Km values were altered by the substitution of amino acid 105 (Johansson et al., 1998). In hen egg-white lysozyme, catalytic activity was decreased, in contrast to our results, depending on the increase in the volume of the side chain of residue15 (Imoto et al., 1994
). The mode of the effect of the residue volume which we found may be applicable only when the rate-limiting step is binding/release of substrates. The effect of the volume may be positive or negative depending on the optimum volume for binding substrates. If the optimum binding is achieved by the residue with smaller volume, increased volume is expected to induce less fitting and induce high kcat, as we found. In contrast, if the optimum binding is achieved by the residue with larger volume, increased volume is expected to induce better fitting.
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Notes |
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5 To whom correspondence should be addressed. E-mail: yamagish{at}ls.toyaku.ac.jp
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Acknowledgments |
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Received November 29, 2001; revised February 12, 2002; accepted February 14, 2002.