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3 Isopropylmalate Dehydrogenase

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Tairo Oshima – 1st expert on this subject based on the ideXlab platform

  • a stable intermediate in the thermal unfolding process of a chimeric 3 Isopropylmalate Dehydrogenase between a thermophilic and a mesophilic enzymes
    Protein Science, 2008
    Co-Authors: Akihiko Yamagishi, Nobuo Tanaka, Yoko Hayashiiwasaki, Masahiro Sakurai, Koichi Numata, Katsuhide Yutani, Tairo Oshima

    Abstract:

    The thermal unfolding process of a chimeric 3Isopropylmalate 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.

  • domain motion in 3 Isopropylmalate Dehydrogenase a strategy to enhance its thermal stability
    Journal of Mathematical and Fundamental Sciences, 2003
    Co-Authors: Zeily Nurachman, Tairo Oshima, Nobuo Tanaka

    Abstract:

    In order to elucidate the thermal properties of Thermus thermophilus 3Isopropylmalate 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 3Isopropylmalate Dehydrogenase.

  • cold adaptation mechanism of mutant enzymes of 3 Isopropylmalate Dehydrogenase from thermus thermophilus
    Protein Engineering, 2002
    Co-Authors: Toshiharu Suzuki, Tairo Oshima, Masako Yasugi, Fumio Arisaka, Akihiko Yamagishi

    Abstract:

    : Random mutagenesis of Thermus thermophilus 3Isopropylmalate 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 3Isopropylmalate 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.

Akihiko Yamagishi – 2nd expert on this subject based on the ideXlab platform

  • a stable intermediate in the thermal unfolding process of a chimeric 3 Isopropylmalate Dehydrogenase between a thermophilic and a mesophilic enzymes
    Protein Science, 2008
    Co-Authors: Akihiko Yamagishi, Nobuo Tanaka, Yoko Hayashiiwasaki, Masahiro Sakurai, Koichi Numata, Katsuhide Yutani, Tairo Oshima

    Abstract:

    The thermal unfolding process of a chimeric 3Isopropylmalate 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.

  • random mutagenesis improves the low temperature activity of the tetrameric 3 Isopropylmalate Dehydrogenase from the hyperthermophile sulfolobus tokodaii
    Protein Engineering Design & Selection, 2008
    Co-Authors: Michika Sasaki, Satoshi Akanuma, Akihiko Yamagishi

    Abstract:

    : 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) 3Isopropylmalate 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.

  • the effects of mutations at position 253 on the thermostability of the bacillus subtilis 3 Isopropylmalate Dehydrogenase subunit interface
    Journal of Biochemistry, 2007
    Co-Authors: Takatoshi Ohkuri, Akihiko Yamagishi

    Abstract:

    3Isopropylmalate 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.

Nobuo Tanaka – 3rd expert on this subject based on the ideXlab platform

  • Design, X-ray crystallography, molecular modelling and thermal stability studies of mutant enzymes at site 172 of 3Isopropylmalate Dehydrogenase from Thermus thermophilus.
    Acta crystallographica. Section D Biological crystallography, 2020
    Co-Authors: C Qu, Nobuo Tanaka, S Akanuma, H Moriyama, T Oshima

    Abstract:

    The relationship between the structure and the thermostability of the 3Isopropylmalate 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.

  • a stable intermediate in the thermal unfolding process of a chimeric 3 Isopropylmalate Dehydrogenase between a thermophilic and a mesophilic enzymes
    Protein Science, 2008
    Co-Authors: Akihiko Yamagishi, Nobuo Tanaka, Yoko Hayashiiwasaki, Masahiro Sakurai, Koichi Numata, Katsuhide Yutani, Tairo Oshima

    Abstract:

    The thermal unfolding process of a chimeric 3Isopropylmalate 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.

  • domain motion in 3 Isopropylmalate Dehydrogenase a strategy to enhance its thermal stability
    Journal of Mathematical and Fundamental Sciences, 2003
    Co-Authors: Zeily Nurachman, Tairo Oshima, Nobuo Tanaka

    Abstract:

    In order to elucidate the thermal properties of Thermus thermophilus 3Isopropylmalate 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 3Isopropylmalate Dehydrogenase.