Crystal structures of mutants of Thermus thermophilus IPMDH adapted to low temperatures

Raita Hirose1, Toshiharu Suzuki1,2, Hideaki Moriyama1,3, Takao Sato1, Akihiko Yamagishi4, Tairo Oshima4 and Nobuo Tanaka1,5

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 14–32–1, Hachioji,Tokyo 192-0392, Japan


    Abstract
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 Abstract
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 Materials and methods
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Random mutagenesis on thermophilic 3-isopropylmalate dehydrogenases (IPMDH; EC 1.1.1.85) produced mutant enzymes which adapt to low temperatures. These mutants had higher activity at lower temperatures than the wild-type enzyme without losing high thermostability. Here we report three structures of the mutants of Thermus thermophilus IPMDH determined by X-ray diffraction which was adapted to a low-temperature environment. Two of them have unstable coenzyme binding states and the other one probably has a stable substrate binding state. The present research suggests that the adaptation is correlated with the binding of either coenzyme or the substrate.

Keywords: cold-adapted mutants/3-isopropylmalate dehydro-genase/thermal stability/Thermus thermophilus/structural analysis


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A thermophilic enzyme is stable and fully active at elevated temperatures. Because the activity of such an enzyme depends on the flexibility of each molecule that comprises it (Vihinen, 1987Go), improved conformational rigidity is indispensable for enhancing the stability of an enzyme against heat denaturation (Zavodszky et al., 1998Go). Many comparisons between thermostable and thermolabile enzymes have been reported in order to elucidate the relationship between their structure and thermostability. These studies have shown that the replacements of enzyme residues result in several stabilizing effects (hydrogen bonds, salt bridges, hydrophobic interactions, etc.). Although these effects stiffen the structure of thermophilic enzymes, they also prevent the enzymes from fitting into a substrate at low temperatures. In other words, the cold-adapted enzymes have highly flexible structures which provide enhanced abilities to undergo conformational changes during catalysis at the cost of thermal stability (Margesin and Schinner, 1994Go; Feller and Gerday, 1997Go).

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, 1994Go; Kadono et al., 1995Go) 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., 1998Go) complexed with IPM revealed the rearrangement of the domains so as to form closed conformations.

Protein engineering (Ulmer, 1983Go) 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 structure–thermostability 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 1Go) 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 IGo). 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, 1993Go) 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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Fig. 1. Structure of the wild-type TtIPMDH monomer. The loop regions and the binding sites of NAD+ and IPM are shown with mutations sites of the cold-adapted mutants. This figure was drawn with MOLSCRIPT (Kraulis, 1991Go).

 

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Table I. Kinetic and thermodynamic parameters at 40°C
 
In the present research, three mutants of TtIPMDH were determined to discuss the adaptation to low temperatures in relation to their three-dimensional structural changes.


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Crystallization and data collection

Each mutant enzyme was purified in the same way as the wild-type enzyme (Yamada et al., 1990Go). For crystallization, the protein solution was concentrated to 15 mg/ml and mixed with an equivalent volume of a reservoir solution of 1.0–1.5 M ammonium sulfate in sodium phosphate buffer (pH 7.0–7.5). Mutant enzymes were crystallized by the hanging drop vapor diffusion method under conditions similar to those for the wild-type enzyme (Imada et al., 1991Go). 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, 1994Go). The diffraction data are summarized in Table IIGo.


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Table II. Collected data
 
Structure determination

All structures were determined using the molecular replacement method with the program AMoRe (Navaza, 1994Go). 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, 1992Go) were applied by AMoRe. Mutations were introduced by examining the electron density map (Figure 2Go) on the display of the program XFIT in the XtalView package (MacRee, 1999Go). The structure of each mutant was refined using the maximum likelihood refinement of REFMAC (Murshudov et al., 1997Go) and validated by PROCHECK (Laskowski et al., 1993Go). Ten percent of the measured reflections were used as free-R test sets (Brunger, 1992Go) in each refinement. The refinement statistics are given in Table IIIGo.



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Fig. 2. FoFc maps calculated omitting replaced residues. The mutant enzyme is drawn in black and wild-type is in gray. (a) V15I; (b) V126M; (c) S92F. Figures 2–4GoGoGo were made using XtalView (MacRee, 1999Go) and Raster3D (Merrit and Bacon, 1997Go) software.

 

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Table III. Final refinement data
 
Modeling

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. 1983Go) 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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The overall structure of each mutant enzyme is similar to that of the wild-type enzyme. It is composed of two identical subunits and each subunit forms an open conformation between the two domains. However, minor structural changes were detected in relation to the adaptation to lower temperatures.

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 (250–254th, Figure 1Go), which is at the interface between the domains, shifts towards the inside of the second domain (Figure 3Go) 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, 1994Go; Kadono et al., 1995Go), 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 IGo).



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Fig. 3. Movement of inter-domain loop of V126M mutant (black) and the wild-type enzyme (gray). The loop is shifted towards the left side (domain 2).

 
In the case of S92F, no significant structural changes were found by the mutation, because the mutated residue is on the molecular surface and does not conflict with the neighboring residues. Although Phe92 is close to the IPM binding site, the kinetic constants indicate that there is a slight change in its affinity for IPM. The free enzyme takes an open conformation with a wide mouth between domains, although the active form of the enzyme is thought to be a closed conformation as found in the crystal structure of the ES complex of TfIPMDH (Imada et al., 1998Go). The effects of the mutation on the active form were speculated upon by constructing a model structure by energy minimization using CHARMm software. The calculations were carried out for both the wild-type enzyme and S92F. The results suggested that the residues from 138 to 141 (Figure 1Go) are shifted towards IPM by 1.6 Å (Figure 4Go), which may be caused by the interaction among aromatic groups, Phe53, Phe41, Phe92, Tyr139 and Phe140. In the model, the binding of IPM seems to be stabilized by Tyr139 approaching IPM.



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Fig. 4. Comparison of closed model of S92F mutant (black) with the open conformation of wild-type enzyme (gray). The aromatic residues are distributed around the Phe92.

 

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Structural shifts

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 (T1/2), in fact the values of Tm and T1/2 are not raised (Table IVGo). 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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Table IV. Thermostability
 
In the case of the Km leading mutant, a large hydrophobic residue, Phe92, is exposed to the solvent and a hydrogen bond between Ser92 and Thr88 found in the wild-type enzyme disappeared. These unfavorable structural changes are compensated for by stabilizing the closed conformation. Previous structural research on TtIPMDH and its homologous enzyme isocitrate dehydrogenase (Kadono et al., 1995Go; Zhang and Koshland, 1995Go) suggested that the active form adopts a closed conformation between domains in order to bind IPM in the crevice of the molecule. The structure shows that the binding of NAD+ is also stabilized in the closed conformation. Accounting for the equilibrium between the open and closed conformations, the mutation may contribute to an increase in the proportion of closed conformations, by which the molecule buries Phe92 within a hydrophobic core. In other words, the enzyme is adapted to low temperature by improvement of the binding of NAD+ and IPM in a closed conformation. The mutation closes the wide mouth between two domains into a closed conformation. However, the thermostability of the mutant enzyme is not improved because the molecule takes an open conformation in solution which is less stable at higher temperature owing to Phe92 protruding into the solvent region.

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., 1997Go), 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., 1996Go). 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., 1995Go), 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 {alpha}-amylase (Aghajari et al., 1998Go). 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., 1997Go), 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.


    Notes
 
2 Present address: Japan Synchrotron Radiation Research Institute (JASRI), 1–1–1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5198, Japan Back

3 Present address: Chemical Resource Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan Back

5 To whom correspondence should be addressed. E-mail: ntanaka{at}bio.titech.ac.jp Back


    Acknowledgments
 
We thank Dr Masako Yasugi for providing information on mutant IPMDH.


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 Materials and methods
 Results
 Discussion
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Received August 23, 2000; revised November 9, 2000; accepted November 15, 2000.