Studies on interdomain interaction of 3-isopropylmalate dehydrogenase froman extreme thermophile, Thermus thermophilus, by constructing chimeric enzymes

Tn our previous study, we showed that a chimeric isopropylmalate dehydrogen ase, 2T2M6T, between an extreme thermophile, Thermus thermophilus, and a me sophile, Bacillus subtilis, isopropylmalate dehydrogenases (the name roughl y denotes the primary structure; the first 20% from the N-terminal is coded by the thermophile leuB gene, next 20% by mesophile, and the rest by the t hermophile gene) denatured in two steps with a stable intermediate, suggest ing that in the chimera some of the interdomain interaction was lost by ami no acid substitutions in the "2M" part. To identify the residues involved i n the interdomain interactions, the first and the second halves of the 2M p art of the chimera were substituted with the corresponding sequence of the thermophile enzyme. Both chimeras. 3T1M6T and 2T1M7T, apparently showed one transition in the thermal denaturation without any stable intermediate sta te, suggesting that the cooperativity of the conformational stability was a t least partly restored by the substitutions. The present study also sugges ted involvement of one or more basic residues in the unusual stability of t he thermophile enzyme.