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Tairo Oshima - One of the best experts on this subject based on the ideXlab platform.
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a stable intermediate in the thermal unfolding process of a chimeric 3 Isopropylmalate Dehydrogenase between a thermophilic and a mesophilic enzymes
Protein Science, 2008Co-Authors: Yoko Hayashiiwasaki, Akihiko Yamagishi, Nobuo Tanaka, Masahiro Sakurai, Koichi Numata, Katsuhide Yutani, Tairo OshimaAbstract:The thermal unfolding process of a chimeric 3-Isopropylmalate Dehydrogenase made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, enzymes was studied by CD spectrophotometry and differential scanning calorimetry (DSC). The enzyme is a homodimer with a subunit containing two structural domains. The DSC melting profile of the chimeric enzyme in 20 mM NaHCO3, pH 10.4, showed two endothermic peaks, whereas that of the T. thermophilus wild-type enzyme had one peak. The CD melting profiles of the chimeric enzyme under the same conditions as the DSC measurement, also indicated biphasic unfolding transition. Concentration dependence of the unfolding profile revealed that the first phase was protein concentration-independent, whereas the second transition was protein concentration-dependent. When cooled after the first transition, the intermediate was isolated, which showed only the second transition upon heating. These results indicated the existence of a stable dimeric intermediate followed by the further unfolding and dissociation in the thermal unfolding of the chimeric enzyme at pH 10-11. Because the portion derived from the mesophilic Isopropylmalate Dehydrogenase in the chimeric enzyme is located in the hinge region between two domains of the enzyme, it is probably responsible for weakening of the interdomain interaction and causing the decooperativity of two domains. The dimeric form of the intermediate suggested that the first unfolding transition corresponds to the unfolding of domain 1 containing the N- and C-termini of the enzyme, and the second to that of domain 2 containing the subunit interface.
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domain motion in 3 Isopropylmalate Dehydrogenase a strategy to enhance its thermal stability
Journal of Mathematical and Fundamental Sciences, 2003Co-Authors: Zeily Nurachman, Tairo Oshima, Nobuo TanakaAbstract:In order to elucidate the thermal properties of Thermus thermophilus 3-Isopropylmalate Dehydrogenase, mutant structures with mutations at the C- terminus were compared with each other. The structural movement can be anticipated from the structural changes among mutants in regions of a minor groove and pillar. Our previous studies revealed that the open-close movement of the active site groove antagonizes to that of the minor groove (like a paperclip) and the thermostability of the enzyme increases when the active site groove is closed. In the present study, it is shown that the motion of the enzyme mainly occurs in the first domain and strand D in the pillar structure is a hinge- bending region of the movement. The motion of the first domain to expand the minor groove may close the active site groove suggesting a mechanism for the enhanced thermal stability of 3-Isopropylmalate Dehydrogenase.
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cold adaptation mechanism of mutant enzymes of 3 Isopropylmalate Dehydrogenase from thermus thermophilus
Protein Engineering, 2002Co-Authors: Toshiharu Suzuki, Tairo Oshima, Masako Yasugi, Fumio Arisaka, Akihiko YamagishiAbstract:: Random mutagenesis of Thermus thermophilus 3-Isopropylmalate Dehydrogenase revealed that a substitution of Val126Met in a hinge region caused a marked increase in specific activity, particularly at low temperatures, although the site is far from the binding residues for 3-Isopropylmalate and NAD. To understand the molecular mechanism, residue 126 was substituted with one of eight other residues, Gly, Ala, Ser, Thr, Glu, Leu, Ile or Phe. Circular dichroism analyses revealed a decreased thermal stability of the mutants (Delta T ((1/2))= 0-13 degrees C), indicating structural perturbations caused by steric conflict with surrounding residues having larger side chains. Kinetic parameters, k(cat) and K(m) values for Isopropylmalate and NAD, were also affected by the mutation, but the resulting k(cat)/K(m) values were similar to that of the wild-type enzyme, suggesting that the change in the catalytic property is caused by the change in free-energy level of the Michaelis complex state relative to that of the initial state. The kinetic parameters and activation enthalpy change (Delta H (double dagger)) showed good correlation with the van der Waals volume of residue 126. These results suggested that the artificial cold adaptation (enhancement of k(cat) value at low temperatures) resulted from the destabilization of the ternary complex caused by the increase in the volume of the residue at position 126.
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high thermal stability of 3 Isopropylmalate Dehydrogenase from thermus thermophilus resulting from low δδcp of unfolding
Protein Engineering, 2001Co-Authors: Chie Motono, Tairo Oshima, Akihiko YamagishiAbstract:To characterize the thermal stability of 3-Isopropylmalate Dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, urea-induced unfolding of the enzyme and of its mesophilic counterpart from Escherichia coli was investigated at various temperatures. The unfolding curves were analyzed with a three-state model for E.coli IPMDH and with a two-state model for T.thermophilus IPMDH, to obtain the free energy change ∆G° of each unfolding process. Other thermodynamic parameters, enthalpy change ∆H, entropy change ∆S and heat capacity change ∆C p, were derived from the temperature dependence of ∆G°. The main feature of the thermophilic enzyme was its lower dependence of ∆G °o n temperature resulting from a low ∆Cp. The thermophilic IPMDH had a significantly lower ∆Cp, 1.73 kcal/mol.K, than that of E.coli IPMDH (20.7 kcal/mol.K). The low ∆Cp of T.thermophilus IPMDH could not be predicted from its change in solvent-accessible surface area ∆ASA. The results suggested that there is a large structural difference between the unfolded state of T.thermophilus and that of E.coli IPMDH. Another responsible factor for the higher thermal stability of T.thermophilus IPMDH was the increase in the most stable temperature Ts. The ∆G° maximum of T.thermophilus IPMDH was much smaller than that of E.coli IPMDH. The present results clearly demonstrated that a higher melting temperature Tm is not necessarily accompanied by a higher ∆G° maximum.
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analysis of the effect of accumulation of amino acid replacements on activity of 3 Isopropylmalate Dehydrogenase from thermus thermophilus
Protein Engineering, 2001Co-Authors: Masako Yasugi, Toshiharu Suzuki, Akihiko Yamagishi, Tairo OshimaAbstract:: 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 k(cat) with a simultaneous increase of K(m) for the coenzyme NAD(+). k(cat) was improved by a decrease in the activation enthalpy, DeltaH( not equal). 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.
Akihiko Yamagishi - One of the best experts on this subject based on the ideXlab platform.
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a stable intermediate in the thermal unfolding process of a chimeric 3 Isopropylmalate Dehydrogenase between a thermophilic and a mesophilic enzymes
Protein Science, 2008Co-Authors: Yoko Hayashiiwasaki, Akihiko Yamagishi, Nobuo Tanaka, Masahiro Sakurai, Koichi Numata, Katsuhide Yutani, Tairo OshimaAbstract:The thermal unfolding process of a chimeric 3-Isopropylmalate Dehydrogenase made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, enzymes was studied by CD spectrophotometry and differential scanning calorimetry (DSC). The enzyme is a homodimer with a subunit containing two structural domains. The DSC melting profile of the chimeric enzyme in 20 mM NaHCO3, pH 10.4, showed two endothermic peaks, whereas that of the T. thermophilus wild-type enzyme had one peak. The CD melting profiles of the chimeric enzyme under the same conditions as the DSC measurement, also indicated biphasic unfolding transition. Concentration dependence of the unfolding profile revealed that the first phase was protein concentration-independent, whereas the second transition was protein concentration-dependent. When cooled after the first transition, the intermediate was isolated, which showed only the second transition upon heating. These results indicated the existence of a stable dimeric intermediate followed by the further unfolding and dissociation in the thermal unfolding of the chimeric enzyme at pH 10-11. Because the portion derived from the mesophilic Isopropylmalate Dehydrogenase in the chimeric enzyme is located in the hinge region between two domains of the enzyme, it is probably responsible for weakening of the interdomain interaction and causing the decooperativity of two domains. The dimeric form of the intermediate suggested that the first unfolding transition corresponds to the unfolding of domain 1 containing the N- and C-termini of the enzyme, and the second to that of domain 2 containing the subunit interface.
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random mutagenesis improves the low temperature activity of the tetrameric 3 Isopropylmalate Dehydrogenase from the hyperthermophile sulfolobus tokodaii
Protein Engineering Design & Selection, 2008Co-Authors: Michika Sasaki, Satoshi Akanuma, Akihiko YamagishiAbstract:: 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 K(m) values for NAD (nicotinamide adenine dinucleotide) and/or the k(cat) 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.
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the effects of mutations at position 253 on the thermostability of the bacillus subtilis 3 Isopropylmalate Dehydrogenase subunit interface
Journal of Biochemistry, 2007Co-Authors: Takatoshi Ohkuri, Akihiko YamagishiAbstract:3-Isopropylmalate Dehydrogenase (IPMDH) is a dimeric enzyme with a strongly hydrophobic core that is composed of residues from four α-helices. We replaced Glu253, which is found in the hydrophobic core and is part of the subunit interface of the Bacillus subtilis (Bs) IPMDH, with several other amino acids to probe. The thermostabilities of the mutants were assessed by measuring the residual enzymatic activities at 40°C after heat treatment and by monitoring changes in ellipticity at 222 nm as the environmental temperature increased incrementally. The results of these studies indicate that, for residues with non-polar side chains, when positioned at residue 253, the thermostabilities of their corresponding mutants correlate positively with the relative hydrophobicities of the side chains. Relative activities of all mutants are lower than that of the wild-type enzyme. For two of the mutants, we directly show that the substitution at position 253 negatively affects Mn 2+ binding, which is required for catalysis. When a lysine is the position 253 residue, the protein dissociates. The results presented herein increase our understanding of the role played by the BsIPMDH dimer interface on the stability and activity of BsIPMDH.
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the effects of multiple ancestral residues on the thermus thermophilus 3 Isopropylmalate Dehydrogenase
FEBS Letters, 2006Co-Authors: Keiko Watanabe, Akihiko YamagishiAbstract:Previously, we showed that mutants of Thermus thermophilus 3-Isopropylmalate Dehydrogenase (IPMDH) each containing a residue (ancestral residue) that had been predicted to exist in a postulated common ancestor protein often have greater thermal stabilities than does the contemporary wild-type enzyme. In this study, the combined effects of multiple ancestral residues were analyzed. Two mutants, containing multiple mutations, Sup3mut (Val181Thr/Pro324Thr/Ala335Glu) and Sup4mut (Leu134Asn/Val181Thr/Pro324Thr/Ala335Glu) were constructed and show greater thermal stabilities than the wild-type and single-point mutant IPMDHs do. Most of the mutants have similar or improved catalytic efficiencies at 70 °C when compared with the wild-type IPMDH.
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designing thermostable proteins ancestral mutants of 3 Isopropylmalate Dehydrogenase designed by using a phylogenetic tree
Journal of Molecular Biology, 2006Co-Authors: Keiko Watanabe, Takatoshi Ohkuri, Shinichi Yokobori, Akihiko YamagishiAbstract:We have recently developed a new method for designing thermostable proteins using phylogenetic trees of enzymes. In this study, we investigated a method for designing proteins with improved stability using 3-Isopropylmalate Dehydrogenase (IPMDH) from Thermus thermophilus as a model enzyme. We designed 12 mutant enzymes, each having an ancestral amino acid residue that was present in the common ancestor of Bacteria and Archaea. At least six of the 12 ancestral mutants tested showed thermal stability higher than that of the original enzyme. The results supported the hyperthermophilic universal ancestor hypothesis. The effect of ancestral residues on IPMDHs of several organisms and on the related enzyme isocitrate Dehydrogenase was summarised and analysed. The effect of an ancestral residue on thermostability did not depend on the degree of conservation of the residue at the site, suggesting that the stabilisation of these mutant proteins is not related to sequence conservation but to the antiquity of the introduced residues. The results suggest also that this method could be an efficient way of designing mutant enzymes with higher thermostability based only on the primary structure and a phylogenetic tree.
Nobuo Tanaka - One of the best experts on this subject based on the ideXlab platform.
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Design, X-ray crystallography, molecular modelling and thermal stability studies of mutant enzymes at site 172 of 3-Isopropylmalate Dehydrogenase from Thermus thermophilus.
Acta crystallographica. Section D Biological crystallography, 2020Co-Authors: C Qu, Nobuo Tanaka, S Akanuma, H Moriyama, T OshimaAbstract:The relationship between the structure and the thermostability of the 3-Isopropylmalate Dehydrogenase from Thermus thermophilus was studied by site-directed mutation of a single Ala residue located at the domain interface. The crystal structures of three mutant enzymes, replacing Ala172 with Gly, Val and Phe, were successfully determined at 2.3, 2.2 and 2.5 A resolution, respectively. Substitution of Ala172 by relatively 'short' residues (Gly, Val or Ile) enlarges or narrows the cavity in the vicinity of the C(beta) atom of Ala172 and the thermostablity of the enzyme shows a good correlation with the hydrophobicity of the substituted residues. Substitution of Ala172 by the 'longer' residues Leu or Phe causes a rearrangement of the domain structure, which leads to a higher thermostability of the enzymes than that expected from the hydrophobicity of the substituted residues. Mutation of Ala172 to negatively charged residues gave an unexpected result: the melting temperature of the Asp mutant enzyme was reduced by 2.7 K while that of the Glu mutant increased by 1.8 K. Molecular-modelling studies indicated that the glutamate side chain was sufficiently long that it did not act as a buried charge as did the aspartate, but instead protruded to the outside of the hydrophobic cavity and contributed to the stability of the enzyme by enhancing the packing of the local side chains and forming an extra salt bridge with the side chain of Lys175.
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a stable intermediate in the thermal unfolding process of a chimeric 3 Isopropylmalate Dehydrogenase between a thermophilic and a mesophilic enzymes
Protein Science, 2008Co-Authors: Yoko Hayashiiwasaki, Akihiko Yamagishi, Nobuo Tanaka, Masahiro Sakurai, Koichi Numata, Katsuhide Yutani, Tairo OshimaAbstract:The thermal unfolding process of a chimeric 3-Isopropylmalate Dehydrogenase made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, enzymes was studied by CD spectrophotometry and differential scanning calorimetry (DSC). The enzyme is a homodimer with a subunit containing two structural domains. The DSC melting profile of the chimeric enzyme in 20 mM NaHCO3, pH 10.4, showed two endothermic peaks, whereas that of the T. thermophilus wild-type enzyme had one peak. The CD melting profiles of the chimeric enzyme under the same conditions as the DSC measurement, also indicated biphasic unfolding transition. Concentration dependence of the unfolding profile revealed that the first phase was protein concentration-independent, whereas the second transition was protein concentration-dependent. When cooled after the first transition, the intermediate was isolated, which showed only the second transition upon heating. These results indicated the existence of a stable dimeric intermediate followed by the further unfolding and dissociation in the thermal unfolding of the chimeric enzyme at pH 10-11. Because the portion derived from the mesophilic Isopropylmalate Dehydrogenase in the chimeric enzyme is located in the hinge region between two domains of the enzyme, it is probably responsible for weakening of the interdomain interaction and causing the decooperativity of two domains. The dimeric form of the intermediate suggested that the first unfolding transition corresponds to the unfolding of domain 1 containing the N- and C-termini of the enzyme, and the second to that of domain 2 containing the subunit interface.
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domain motion in 3 Isopropylmalate Dehydrogenase a strategy to enhance its thermal stability
Journal of Mathematical and Fundamental Sciences, 2003Co-Authors: Zeily Nurachman, Tairo Oshima, Nobuo TanakaAbstract:In order to elucidate the thermal properties of Thermus thermophilus 3-Isopropylmalate Dehydrogenase, mutant structures with mutations at the C- terminus were compared with each other. The structural movement can be anticipated from the structural changes among mutants in regions of a minor groove and pillar. Our previous studies revealed that the open-close movement of the active site groove antagonizes to that of the minor groove (like a paperclip) and the thermostability of the enzyme increases when the active site groove is closed. In the present study, it is shown that the motion of the enzyme mainly occurs in the first domain and strand D in the pillar structure is a hinge- bending region of the movement. The motion of the first domain to expand the minor groove may close the active site groove suggesting a mechanism for the enhanced thermal stability of 3-Isopropylmalate Dehydrogenase.
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design x ray crystallography molecular modelling and thermal stability studies of mutant enzymes at site 172 of 3 Isopropylmalate Dehydrogenase from thermus thermophilus
Acta Crystallographica Section D-biological Crystallography, 2001Co-Authors: Chunxu Qu, Nobuo Tanaka, Hideaki Moriyama, Satoshi Akanuma, Tairo OshimaAbstract:The relationship between the structure and the thermostability of the 3-Isopropylmalate Dehydrogenase from Thermus thermophilus was studied by site-directed mutation of a single Ala residue located at the domain interface. The crystal structures of three mutant enzymes, replacing Ala172 with Gly, Val and Phe, were successfully determined at 2.3, 2.2 and 2.5 A resolution, respectively. Substitution of Ala172 by relatively `short' residues (Gly, Val or Ile) enlarges or narrows the cavity in the vicinity of the Cβ atom of Ala172 and the thermostablity of the enzyme shows a good correlation with the hydrophobicity of the substituted residues. Substitution of Ala172 by the `longer' residues Leu or Phe causes a rearrangement of the domain structure, which leads to a higher thermostability of the enzymes than that expected from the hydrophobicity of the substituted residues. Mutation of Ala172 to negatively charged residues gave an unexpected result: the melting temperature of the Asp mutant enzyme was reduced by 2.7 K while that of the Glu mutant increased by 1.8 K. Molecular-modelling studies indicated that the glutamate side chain was sufficiently long that it did not act as a buried charge as did the aspartate, but instead protruded to the outside of the hydrophobic cavity and contributed to the stability of the enzyme by enhancing the packing of the local side chains and forming an extra salt bridge with the side chain of Lys175.
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the initial step of the thermal unfolding of 3 Isopropylmalate Dehydrogenase detected by the temperature jump laue method
Protein Engineering, 2000Co-Authors: Tetsuya Hori, Tairo Oshima, Hideaki Moriyama, Jitsutaro Kawaguchi, Yoko Hayashiiwasaki, Nobuo TanakaAbstract:: A temperature-jump (T-jump) time-resolved X-ray crystallographic technique using the Laue method was developed to detect small, localized structural changes of proteins in crystals exposed to a temperature increase induced by laser irradiation. In a chimeric protein between thermophilic and mesophilic 3-Isopropylmalate Dehydrogenases (2T2M6T), the initial structural change upon T-jump to a denaturing temperature (approximately 90 degrees C) was found to be localized at a region which includes a beta-turn and a loop located between the two domains of the enzyme. A mutant, 2T2M6T-E110P/S111G/S113E, having amino acid replacements in this beta-turn region with the corresponding residues of the thermophilic enzyme, showed greater stability than the original chimera (increase of T:(m) by approximately 10 degrees C) and no T-jump-induced structural change in this region was detected by our method. These results indicate that thermal unfolding of the original chimeric enzyme, 2T2M6T, is triggered in this beta-turn region.
Satoshi Akanuma - One of the best experts on this subject based on the ideXlab platform.
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random mutagenesis improves the low temperature activity of the tetrameric 3 Isopropylmalate Dehydrogenase from the hyperthermophile sulfolobus tokodaii
Protein Engineering Design & Selection, 2008Co-Authors: Michika Sasaki, Satoshi Akanuma, Akihiko YamagishiAbstract:: 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 K(m) values for NAD (nicotinamide adenine dinucleotide) and/or the k(cat) 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.
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design x ray crystallography molecular modelling and thermal stability studies of mutant enzymes at site 172 of 3 Isopropylmalate Dehydrogenase from thermus thermophilus
Acta Crystallographica Section D-biological Crystallography, 2001Co-Authors: Chunxu Qu, Nobuo Tanaka, Hideaki Moriyama, Satoshi Akanuma, Tairo OshimaAbstract:The relationship between the structure and the thermostability of the 3-Isopropylmalate Dehydrogenase from Thermus thermophilus was studied by site-directed mutation of a single Ala residue located at the domain interface. The crystal structures of three mutant enzymes, replacing Ala172 with Gly, Val and Phe, were successfully determined at 2.3, 2.2 and 2.5 A resolution, respectively. Substitution of Ala172 by relatively `short' residues (Gly, Val or Ile) enlarges or narrows the cavity in the vicinity of the Cβ atom of Ala172 and the thermostablity of the enzyme shows a good correlation with the hydrophobicity of the substituted residues. Substitution of Ala172 by the `longer' residues Leu or Phe causes a rearrangement of the domain structure, which leads to a higher thermostability of the enzymes than that expected from the hydrophobicity of the substituted residues. Mutation of Ala172 to negatively charged residues gave an unexpected result: the melting temperature of the Asp mutant enzyme was reduced by 2.7 K while that of the Glu mutant increased by 1.8 K. Molecular-modelling studies indicated that the glutamate side chain was sufficiently long that it did not act as a buried charge as did the aspartate, but instead protruded to the outside of the hydrophobic cavity and contributed to the stability of the enzyme by enhancing the packing of the local side chains and forming an extra salt bridge with the side chain of Lys175.
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further improvement of the thermal stability of a partially stabilized bacillus subtilis 3 Isopropylmalate Dehydrogenase variant by random and site directed mutagenesis
FEBS Journal, 1999Co-Authors: Nobuo Tanaka, Akihiko Yamagishi, Satoshi Akanuma, Tairo OshimaAbstract:A thermostabilized mutant of Bacillus subtilis 3-Isopropylmalate Dehydrogenase (IPMDH) obtained in a previous study contained a set of triple amino acid substitutions. To further improve the stability of the mutant, we used a random mutagenesis technique and identified two additional thermostabilizing substitutions, Thr22Lys and Met256Val, that separately endowed the protein with further stability. We introduced the two mutations into a single enzyme molecule, thus constructing a mutant with overall quintuple mutations. Other studies have suggested that an improved hydrophobic subunit interaction and a rigid type II β-turn play important roles in enhancing the protein stability. Based on those observations, we successively introduced amino acid substitutions into the mutant with the quintuple mutations by site-directed mutagenesis: Glu253 at the subunit interface was replaced by Leu to increase the hydrophobic interaction between the subunits; Glu112, Ser113 and Ser115 that were involved in the formation of the turn were replaced by Pro, Gly and Glu, respectively, to make the turn more rigid. The thermal stability of the mutants was determined based on remaining activity after heat treatment and first-order rate constant of thermal unfolding, which showed gradual increases in thermal stability as more mutations were included.
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serial increase in the thermal stability of 3 Isopropylmalate Dehydrogenase from bacillus subtilis by experimental evolution
Protein Science, 1998Co-Authors: Nobuo Tanaka, Akihiko Yamagishi, Satoshi Akanuma, Tairo OshimaAbstract:We improved the thermal stability of 3-Isopropylmalate Dehydrogenase from Bacillus subtilis by an in vivo evolutionary technique using an extreme thermophile, Thermus thermophilus, as a host cell. The leuB gene encoding B. subtilis 3-Isopropylmalate Dehydrogenase was integrated into the chromosome of a leuB-deficient strain of T. thermophilus. The resulting transformant showed a leucine-autotrophy at 56 degrees C but not at 61 degrees C and above. Phenotypically thermostabilized strains that can grow at 61 degrees C without leucine were isolated from spontaneous mutants. Screening temperature was stepwise increased from 61 to 66 and then to 70 degrees C and mutants that showed a leucine-autotrophic growth at 70 degrees C were obtained. DNA sequence analyses of the leuB genes from the mutant strains revealed three stepwise amino acid replacements, threonine-308 to isoleucine, isoleucine-95 to leucine, and methionine-292 to isoleucine. The mutant enzymes with these amino acid replacements were more stable against heat treatment than the wild-type enzyme. Furthermore, the triple-mutant enzyme showed significantly higher specific activity than that of the wild-type enzyme.
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effect of polar side chains at position 172 on thermal stability of 3 Isopropylmalate Dehydrogenase from thermus thermophilus
FEBS Letters, 1997Co-Authors: Nobuo Tanaka, Akihiko Yamagishi, Satoshi Akanuma, Chunxu Qu, Tairo OshimaAbstract:To understand the role of the amino acid residue at position 172 in the conformational stability, four mutant enzymes of Thermus thermophilus 3-Isopropylmalate Dehydrogenase in which Ala172 was replaced with Asp, Glu, Asn, and Gln were prepared by site-directed mutagenesis. Three mutants were more stable than the wild-type enzyme. No significant change in catalytic properties was found in the mutant enzymes. The molecular modeling studies suggested that the enhanced thermostability of the mutant enzymes resulted from the formation of extra electrostatic interactions and/or improvement of hydrophobic packing of the interior core.
Hideaki Moriyama - One of the best experts on this subject based on the ideXlab platform.
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design x ray crystallography molecular modelling and thermal stability studies of mutant enzymes at site 172 of 3 Isopropylmalate Dehydrogenase from thermus thermophilus
Acta Crystallographica Section D-biological Crystallography, 2001Co-Authors: Chunxu Qu, Nobuo Tanaka, Hideaki Moriyama, Satoshi Akanuma, Tairo OshimaAbstract:The relationship between the structure and the thermostability of the 3-Isopropylmalate Dehydrogenase from Thermus thermophilus was studied by site-directed mutation of a single Ala residue located at the domain interface. The crystal structures of three mutant enzymes, replacing Ala172 with Gly, Val and Phe, were successfully determined at 2.3, 2.2 and 2.5 A resolution, respectively. Substitution of Ala172 by relatively `short' residues (Gly, Val or Ile) enlarges or narrows the cavity in the vicinity of the Cβ atom of Ala172 and the thermostablity of the enzyme shows a good correlation with the hydrophobicity of the substituted residues. Substitution of Ala172 by the `longer' residues Leu or Phe causes a rearrangement of the domain structure, which leads to a higher thermostability of the enzymes than that expected from the hydrophobicity of the substituted residues. Mutation of Ala172 to negatively charged residues gave an unexpected result: the melting temperature of the Asp mutant enzyme was reduced by 2.7 K while that of the Glu mutant increased by 1.8 K. Molecular-modelling studies indicated that the glutamate side chain was sufficiently long that it did not act as a buried charge as did the aspartate, but instead protruded to the outside of the hydrophobic cavity and contributed to the stability of the enzyme by enhancing the packing of the local side chains and forming an extra salt bridge with the side chain of Lys175.
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the initial step of the thermal unfolding of 3 Isopropylmalate Dehydrogenase detected by the temperature jump laue method
Protein Engineering, 2000Co-Authors: Tetsuya Hori, Tairo Oshima, Hideaki Moriyama, Jitsutaro Kawaguchi, Yoko Hayashiiwasaki, Nobuo TanakaAbstract:: A temperature-jump (T-jump) time-resolved X-ray crystallographic technique using the Laue method was developed to detect small, localized structural changes of proteins in crystals exposed to a temperature increase induced by laser irradiation. In a chimeric protein between thermophilic and mesophilic 3-Isopropylmalate Dehydrogenases (2T2M6T), the initial structural change upon T-jump to a denaturing temperature (approximately 90 degrees C) was found to be localized at a region which includes a beta-turn and a loop located between the two domains of the enzyme. A mutant, 2T2M6T-E110P/S111G/S113E, having amino acid replacements in this beta-turn region with the corresponding residues of the thermophilic enzyme, showed greater stability than the original chimera (increase of T:(m) by approximately 10 degrees C) and no T-jump-induced structural change in this region was detected by our method. These results indicate that thermal unfolding of the original chimeric enzyme, 2T2M6T, is triggered in this beta-turn region.
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crystallization and preliminary x ray studies on the hyperstable 3 Isopropylmalate Dehydrogenase from the thermoacidophilic archaeon sulfolobus sp strain 7
Acta Crystallographica Section D-biological Crystallography, 1998Co-Authors: Toshiharu Suzuki, Hideaki Moriyama, Nobuo Tanaka, Raita Hirose, Masahiro Sakurai, Tairo OshimaAbstract:3-Isopropylmalate Dehydrogenase from the thermoacidophilic archaeon, Sulfolobus sp. strain 7, has been crystallized by the vapor-diffusion method. The crystals were grown from a solution containing ammonium sulfate, 2-methyl-2,4-pentanediol and magnesium chloride. The crystallization requires 2-methyl-2,4-pentanediol to avoid twinning of the crystals. The crystal belongs to the orthorhombic system with the space group P2221 and unit-cell dimensions a = 67.9, b = 93.3 and c = 134.1 A.
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a mutation at the interface between domains causes rearrangement of domains in 3 Isopropylmalate Dehydrogenase
Protein Engineering, 1997Co-Authors: Chunxu Qu, Nobuo Tanaka, Hideaki Moriyama, Satoshi Akanuma, Tairo OshimaAbstract:: The structure of a thermostable Ala172Leu mutant, designated A172L, of 3-Isopropylmalate Dehydrogenase from Thermus thermophilus was determined. The crystal belongs to space group P2(1), with cell parameters a = 55.5 A, b = 88.1 A, c = 72.0 A and beta = 100.9 degrees. There is one dimer in each asymmetric unit. The final R factor is 17.8% with 69 water molecules at 2.35 A resolution. The mutation is located at the interface between domains and the C alpha trace of the mutant structure deviates from that of the native structure by as much as 1.7 A, while the structure of each domain barely changes. The mutant enzyme has a more closed conformation compared with the wild-type enzyme as a result of the replacement of Ala with Leu at residue 172. These structural variations were found independent of the crystal packing, because the structure of wild type was the same in crystals obtained in different precipitants. The hinge regions for the movement of domains are located around the active cleft of the enzyme, an observation that implies that the mobility of domains around the hinge is indispensable for the activity of the enzyme. The larger side chain at the mutated site contributed to the thermostability of the mutant protein by enhancing the local packing of side chains, and also by shifting the backbone of the opposing domain.
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cryocrystallography of 3 Isopropylmalate Dehydrogenase from thermus thermophilus and its chimeric enzyme
Acta Crystallographica Section D-biological Crystallography, 1996Co-Authors: Chikahiro Nagata, Hideaki Moriyama, Nobuo Tanaka, Masayoshi Nakasako, Masaki Yamamoto, Tatzuo Ueki, Tairo OshimaAbstract:The crystal structures of thermostable enzyme, 3-Isopropylmalate Dehydrogenase of Thermus thermophilus (10T) and a chimeric enzyme between T. thermophilus and Bacillus subtilus with one point mutation (cS82R), were determined at both 100 and 150 K. At the cryogenic condition, the volume of the unit cell decreased by 5% as a result of a contraction in the solvent region. Although the overall structures of both enzymes at low temperature were the same as that of 10T at room temperature, interactions between two domains and between two subunits in a functional dimer of cS82R were significantly altered. The decrease in the average temperature factor of 10T at low temperature and no significant decrease for cS82R suggested that the structure of the engineered enzyme (cS82R) may have many conformational substates even at low temperature, while the native enzyme (10T) at low temperature has a more definite conformation than that at room temperature. The location of water molecules around the enzyme molecule and the calculation of the radii of gyration suggested that cS82R had a weaker hydration than 10T.