TY - JOUR
T1 - Thermostabilization of a chimeric enzyme by residue substitutions
T2 - Four amino acid residues in loop regions are responsible for the thermostability of Thermus thermophilus isopropylmalate dehydrogenase
AU - Numata, Koichi
AU - Hayashi-Iwasaki, Yoko
AU - Kawaguchi, Jitsutaro
AU - Sakurai, Masahiro
AU - Moriyama, Hideaki
AU - Tanaka, Nobuo
AU - Oshima, Tairo
N1 - Funding Information:
The authors are gratefully indebted to Dr. H. Umeyama for the operation of ‘SCROIL’ and Dr. R. Hawkins for critical reading of the manuscript. This investigation was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sport and Culture of Japan (Nos. 02403029 and 04044068).
PY - 2001/2/9
Y1 - 2001/2/9
N2 - A chimeric 3-isopropylmalate dehydrogenase, named 2T2M6T, made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, was found to be considerably more labile than the T. thermophilus wild-type isopropylmalate dehydrogenase. In order to identify the molecular basis of the thermal stability of the T. thermophilus isopropylmalate dehydrogenase, 11 amino acid residues in the mesophilic portion of the chimera were substituted by the corresponding residues of the T. thermophilus enzyme, and the effects of the side chain substitutions were analyzed by comparing the reaction rate of irreversible heat denaturation and catalytic parameters of the mutant chimeras with those of the original chimera, 2T2M6T. Four single-site mutants were successfully stabilized without any loss of the catalytic function. All these four sites are located in loop regions of the enzyme. Our results strongly suggest the importance of these loop structures to the extreme stability of the T. thermophilus isopropylmalate dehydrogenase.
AB - A chimeric 3-isopropylmalate dehydrogenase, named 2T2M6T, made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, was found to be considerably more labile than the T. thermophilus wild-type isopropylmalate dehydrogenase. In order to identify the molecular basis of the thermal stability of the T. thermophilus isopropylmalate dehydrogenase, 11 amino acid residues in the mesophilic portion of the chimera were substituted by the corresponding residues of the T. thermophilus enzyme, and the effects of the side chain substitutions were analyzed by comparing the reaction rate of irreversible heat denaturation and catalytic parameters of the mutant chimeras with those of the original chimera, 2T2M6T. Four single-site mutants were successfully stabilized without any loss of the catalytic function. All these four sites are located in loop regions of the enzyme. Our results strongly suggest the importance of these loop structures to the extreme stability of the T. thermophilus isopropylmalate dehydrogenase.
KW - 3-Isopropylmalate dehydrogenase
KW - Chimera
KW - Protein stability
KW - Site-directed mutagenesis
KW - Thermus thermophilus
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U2 - 10.1016/S0167-4838(00)00275-2
DO - 10.1016/S0167-4838(00)00275-2
M3 - Article
C2 - 11342043
AN - SCOPUS:0035830685
SN - 0167-4838
VL - 1545
SP - 174
EP - 183
JO - Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
JF - Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
IS - 1-2
ER -