title: Mirror image mutations reveal the significance of an intersubunit ion cluster in the stability of 3-isopropylmalate dehydrogenase.
abstract: The comparison of the three-dimensional structures of thermophilic (Thermus thermophilus) and mesophilic (Escherichia coli) 3-isopropylmalate dehydrogenases (IPMDH, EC 1.1.1.85) suggested that the existence of extra ion pairs in the thermophilic enzyme found in the intersubunit region may be an important factor for thermostability. As a test of our assumption, glutamine 200 in the E. coli enzyme was turned into glutamate (Q200E mutant) to mimic the thermophilic enzyme at this site by creating an intersubunit ion pair which can join existing ion clusters. At the same site in the thermophilic enzyme we changed glutamate 190 into glutamine (E190Q), hereby removing the corresponding ion pair. These single amino acid replacements resulted in increased thermostability of the mesophilic and decreased thermostability of the thermophilic enzyme, as measured by spectropolarimetry and differential scanning microcalorimetry.
authors.affiliation: Institute of Enzymology, Hungarian Academy of Sciences, P.O. Box 7, H-1518, Budapest, Hungary.
authors.forename: A
authors.initials: A
authors.lastname: Németh
authors.forename: A
authors.initials: A
authors.lastname: Svingor
authors.forename: M
authors.initials: M
authors.lastname: Pócsik
authors.forename: J
authors.initials: J
authors.lastname: Dobó
authors.forename: C
authors.initials: C
authors.lastname: Magyar
authors.forename: A
authors.initials: A
authors.lastname: Szilágyi
authors.forename: P
authors.initials: P
authors.lastname: Gál
authors.forename: P
authors.initials: P
authors.lastname: Závodszky
mesh_terms: D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics*; D019943:Amino Acid Substitution; D002384:Catalysis; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D007477:Ions; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D007477:Ions; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase