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

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.