Cold adaptation of the thermophilic enzyme 3-isopropylmalate dehydrogenase

We have performed random mutagenesis coupled with selection to isolate mutant enzymes with high catalytic activities at low temperature from thermophilic 3-isopropylmalate dehydrogenase (IPMDH) originally isolated from Thermus thermophilus, Five cold-adapted mutant IPMDHs with single-amino-acid substitutions were obtained and analyzed. Kinetic analysis revealed that there are two types of cold-adapted mutant IPMDH: k(cat)-improved (improved in k(cat)) and K-m-improved (improved in k(cat)/K-m) types. To determine the mechanisms of cold adaptation of these mutants, thermodynamic parameters were estimated and compared with those of the Escherichia coli wild-type IPMDH, The DeltaG(m) values for Michaelis intermediate formation of the k(cat)-improved-type enzymes were larger than that of the T. thermophilus wild-type IPMDH and similar to that of the E. coli wild-type IPMDH. The DeltaG(m) values of K-m-improved-type enzymes were smaller than that of the T. thermophilus wild-type IPMDH, Fitting of NAD(+) binding was improved in the K-m-improved-type enzymes. The two types of cold-adapted mutants employed one of the two strategies of E. coli wild-type IPMDH: relative destabilization of the Michaelis complex in k(cat)-improved-type, and destabilization of the rate-limiting step in K-m-improved type mutants, Some cold-adapted mutant IPMDHs retained thermostability similar to that of the T. thermophilus wild-type IPMDH.