Thermostabilization of a chimeric enzyme by residue substitutions: four amino acid residues in loop regions are responsible for the thermostability of Thermus thermophilus isopropylmalate dehydrogenase
K. Numata et al., Thermostabilization of a chimeric enzyme by residue substitutions: four amino acid residues in loop regions are responsible for the thermostability of Thermus thermophilus isopropylmalate dehydrogenase, BBA-PROT ST, 1545(1-2), 2001, pp. 174-183
Citations number
37
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY
A chimeric 3-isopropylmalate dehydrogenase, named 2T2M6T, made of parts fro
m an extreme thermophile, Thermus thermophilus, and a mesophile, Bavillus s
ubtilis, 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 dehy
drogenase, 11 amino acid residues in the mesophilic portion of the chimera
were substituted by the corresponding residues of the T. thermophilus enzym
e, and the effects of the side chain substitutions were analyzed by compari
ng the reaction rate of irreversible heat denaturation and catalytic parame
ters of the mutant chimeras with those of the original chimera, 2T2M6T. Fou
r 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 structur
es to the extreme stability of the T. thermophilus isopropylmalate dehydrog
enase. (C) 2001 Elsevier Science B.V. All rights reserved.