1 Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501 and 4 School of Life Science, Tokyo University of Pharmacy and Life Science, Horinouchi 14321, Hachioji,Tokyo 192-0392, Japan
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Abstract |
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Keywords: cold-adapted mutants/3-isopropylmalate dehydro-genase/thermal stability/Thermus thermophilus/structural analysis
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Introduction |
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The enzyme 3-isopropylmalate dehydrogenase (IPMDH; EC 1.1.1.85) in the leucine biosynthesis pathway catalyzes the dehydrogenation and decarboxylation of 3-isopropylmalate (IPM). Usually, the enzyme is assembled from two identical subunits, each of which is composed of two domains. The active site is located in a cleft between two domains. The enzyme allows an induced fit upon binding of the substrate IPM and coenzyme NAD+ on the active site. The crystal structure of T.thermophilus IPMDH (TtIPMDH) complexed with NAD+ (Hurley and Dean, 1994; Kadono et al., 1995
) shows that a surface loop hangs over the adenine base of the coenzyme. The crystal structure of IPMDH from Thiobacillus ferrooxidans (TfIPMDH) (Imada et al., 1998
) complexed with IPM revealed the rearrangement of the domains so as to form closed conformations.
Protein engineering (Ulmer, 1983) methods such as site-directed mutagenesis are used to enhance several properties of enzymes. However, protein engineering has a technological limit in enhancing thermostability, since the principle of structurethermostability relationships has not been clarified despite a number of experiments. Evolution engineering, therefore, is an effective method to enhance thermostability. It applies the combination of random mutation and selection, which are important factors in molecular evolutionary mechanisms. Under appropriate conditions, this method can also be applied to adapt the protein to lower temperature and to improve thermostability.
Evolution engineering experiments on TtIPMDH to adapt it to a temperature of 40°C produced mutant enzymes adapted to low temperature (Suzuki et al.). The enzymes are named V15I, V126M and S92F after their mutation sites and residues (Figure 1) and are called cold-adapted mutants hereafter. Two alternative strategies for adaptation are suggested from the fact that the kinetic parameters of S92F differ from those of V15I and V126M (Table I
). The larger Km values of V15I and V126M for NAD+ imply that they release bound NADH easily. Release of the products seems to be a rate-limiting step as in the case of the homologous enzyme isocitrate dehydrogenase (Dean and Koshland, 1993
) and the quick release of bound NADH accelerates catalytic reactions that have large kcat values. S92F has a greater affinity for NAD+ and a higher catalytic efficiency, kcat/Km (NAD+), as a result of a lowering of Km for NAD+ at 40°C. The notable structural peculiarity may be related to the individual method of adaptation.
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Materials and methods |
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Each mutant enzyme was purified in the same way as the wild-type enzyme (Yamada et al., 1990). For crystallization, the protein solution was concentrated to 15 mg/ml and mixed with an equivalent volume of a reservoir solution of 1.01.5 M ammonium sulfate in sodium phosphate buffer (pH 7.07.5). Mutant enzymes were crystallized by the hanging drop vapor diffusion method under conditions similar to those for the wild-type enzyme (Imada et al., 1991
). Drops of mixed solutions were sealed over 1 ml of reservoir solution. After 1 week in an incubator at 25°C, the crystals were grown to a size of ~0.4x0.2x0.2 mm. A series of diffraction images from the crystal sealed in a glass capillary were obtained with the oscillation method on a Rigaku R-AXIS IV X-ray detector at 25°C. The images were converted to intensities using MOSFLM software (Leslie, 1990) and subsequent processing was conducted using CCP4 suite software (Collaborative Computational Project, 1994
). The diffraction data are summarized in Table II
.
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All structures were determined using the molecular replacement method with the program AMoRe (Navaza, 1994). The search model was constructed by removing water molecules from the PDB file of the wild-type enzyme (PDB code: 1IPD). The highest peaks of the rotation and translation functions were selected and rigid body refinements (Castellano and Olivia, 1992
) were applied by AMoRe. Mutations were introduced by examining the electron density map (Figure 2
) on the display of the program XFIT in the XtalView package (MacRee, 1999
). The structure of each mutant was refined using the maximum likelihood refinement of REFMAC (Murshudov et al., 1997
) and validated by PROCHECK (Laskowski et al., 1993
). Ten percent of the measured reflections were used as free-R test sets (Brunger, 1992
) in each refinement. The refinement statistics are given in Table III
.
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Since the ES complex of TtIPMDH took a closed conformation, the structures of the closed conformation of S92F and the wild-type enzyme were simulated with the programs QUANTA and CHARMm. Each domain of S92F was fitted to the domains of the closed conformation of TfIPMDH. Two hundred steps of the Adopted-basis Newton Raphson minimization (Brooks et al. 1983) were applied after adding hydrogen atoms to the model. The same procedure of simulation was applied to the wild-type enzyme to compare with S92F.
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Results |
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In the structure of V15I, the mutation of Val into Ile fits well into the cavity made by surrounding residues, Ile11, Pro271, Ala290 and His273, most of which are hydrophobic and contribute to the hydrophobic interaction. Hence, the replacement to Ile15 narrowed the NAD+ binding cleft by 0.7 Å by forcing His273 upward. These facts imply that NAD+ binding might be weakened; indeed the Km value for NAD+ increased about 6-fold compared with that of the wild-type enzyme.
In V126M, the side chain Met126 also fitted well into the cavity surrounded by hydrophobic residues, Ala106, Val128, Pro227, Ala247 and Leu250. The flexible loop (250254th, Figure 1), which is at the interface between the domains, shifts towards the inside of the second domain (Figure 3
) by the hydrophobic interaction between Met126 and Leu250. Although in the wild-type enzyme the end of the loop, Leu254, moves towards NAD+ when IPM binds to the enzyme (Hurley and Dean, 1994
; Kadono et al., 1995
), the locking of the loop by the replacement to Met126 prevents the move of Leu254 towards NAD+, which may explain the decrease in affinity for NAD+, as indicated by the large Km (Table I
).
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Discussion |
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The kinetic parameters of mutants suggested two different strategies against the lower temperature environment. V15I and V126M are called kcat improved mutants, because of the larger kcat values than for the wild-type enzyme. S92F is called Km improved mutant whose smaller Km value for NAD+ increases the kcat/Km (NAD+) value and a high activity is suggested.
In the case of kcat improved mutants, the replaced residues fill cavities and do not disturb the hydrophobic core. Although it would be expected that increased hydrophobic interaction would stabilize a protein and raise the thermal denaturation temperature (T), in fact the values of Tm and T
are not raised (Table IV
). This implies that the thermostabilities of mutants are not improved, because the weakened bonding of NAD+ contributes in increasing the value of kcat by immediately releasing NADH.
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Mutation sites
It is noteworthy that all mutation sites affect the ligand binding sites indirectly without major structural changes. In the case of the cold-adapted mutant of subtilisin (Kano et al., 1997), the mutation site is on the opposite side of the catalytic triad. Most of the mutation sites that stabilize TtIPMDH are either at the domain interface (Ile172, Kotsuka et al., 1995; Akanuma et al., 1997; Qu et al., 1997) or behind the helix (Ile93, Tamakoshi et al., 1995). These mutation sites are not in the direct vicinity of the binding site. If the mutation occurs in a ligand-binding site, severe distortion of the binding site would occur and alter the binding specificity of the enzyme. For example, mutations on only three NAD+ binding residues of TtIPMDH affected the cofactor binding and decreased the affinity for NAD+ by 1/153 (Chen et al., 1996
). In order to improve the function, it would be appropriate to shift the conformations of side chains involved in the active site rather than to replace the side chains. It is obvious that enzymes have evolved with conservation of the homology of ligand-binding sites among species.
Effects of evolutionary engineering
Generally, the introduction of hydrophobic interaction is used in thermal stabilization of the enzyme. Since the hydrophobic interaction is entropic (Tanford, 1962), the enzyme becomes more stable at high temperatures if hydrophobic interactions are strengthened. In the case of the buried core of lysozyme (Shih et al., 1995), a correlation between thermostability and hydrophobicity of introduced residues in hydrophobic pockets was found. In contrast, it was reported that the exposed hydrophobic residues contribute to a lower stability of cold-adapted
-amylase (Aghajari et al., 1998
). In the case of IPMDH, the thermostabilities of mutants with increased hydrophobic introductions were not improved.
In a sequence alignment of eight mesophilic IPMDH (Wallon et al., 1997), isoleucine at position 15 is found for four species and methionine at 126 and phenylalanine at 92 are not found. Therefore, the present mutations are regarded as not being correlated with the primary sequences of the mesophilic enzymes. In another study, the effects of these mutations were found not to be cumulative (M.Yasugi, personal communication). From these facts, the present mutations may differ from the adaptation on Earth.
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Notes |
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3 Present address: Chemical Resource Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan
5 To whom correspondence should be addressed. E-mail: ntanaka{at}bio.titech.ac.jp
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Acknowledgments |
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References |
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Received August 23, 2000; revised November 9, 2000; accepted November 15, 2000.