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 Delta G(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 Delta G(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.
Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.
M
M
Yasugi
M
M
Amino
T
T
Suzuki
T
T
Oshima
A
A
Yamagishi
D050539:3-Isopropylmalate Dehydrogenase; D000222:Adaptation, Physiological; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D003001:Cloning, Molecular; D003080:Cold Temperature; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase