HYDROPHOBIC INTERACTION AT THE SUBUNIT INTERFACE CONTRIBUTES TO THE THERMOSTABILITY OF 3-ISOPROPYLMALATE DEHYDROGENASE FROM AN EXTREME THERMOPHILE, THERMUS-THERMOPHILUS

We cloned and sequenced the leuB gene encoding 3-isopropylmalate dehyd rogenase from Escherichia coli K-12 (JM103). Errors (33 residues) were found and corrected in the sequence previously reported for the leuB gene of Thermus thermaphilus. The three-dimensional structure of the t hermophile enzyme and the amino acid sequence comparison suggested tha t a part of the high stability of the T. thermophilus enzyme is confer red by increased hydrophobic interaction at the subunit-subunit interf ace. Two residues at the interface of the T. thermophilus enzyme, Leu2 46 and Val249, are substituted with less hydrophobic residues, Glu and Met, respectively, in the E. coli enzyme, whereas other residues in t his region are highly conserved. The mutated T. thermophilus enzyme [L 246E, V249M]IPMDH had reduced stability to heat. Two residues of the E . coli dehydrogenase, Glu256 and Met259, were replaced with the corres ponding residues from the thermophile sequence. The resulted mutant en zyme was more resistant to heat than the wild-type enzyme.