title: Purification, catalytic properties and thermostability of 3-isopropylmalate dehydrogenase from Escherichia coli.
abstract: 3-isopropylmalate dehydrogenase (IPMDH) from Escherichia coli was overexpressed, purified and crystallized. The enzyme was characterized and compared to its thermophilic counterpart from Thermus thermophilus strain HB8. As in the thermophile enzyme, the activity of E. coli IPMDH was dependent on the divalent cations, Mg2+ or Mn2+, with Mn2+ being the preferred cation. Activity was also strongly influenced by KCl: 0.3 M were necessary for the optimal activity. At 40 degrees C the K(m) of E. coli IPMDH was 105 microM for IPM and 321 microM for NAD, the kcat was 69 s-1. The half denaturation temperature was 64 degrees C, which was 20 degrees C lower than that of the thermophile enzyme.
authors.affiliation: Rosenstiel Medical Sciences Research Center, Brandeis University, Waltham, MA 02254-9110, USA.
authors.forename: G
authors.initials: G
authors.lastname: Wallon
authors.forename: K
authors.initials: K
authors.lastname: Yamamoto
authors.forename: H
authors.initials: H
authors.lastname: Kirino
authors.forename: A
authors.initials: A
authors.lastname: Yamagishi
authors.forename: S T
authors.initials: ST
authors.lastname: Lovett
authors.forename: G A
authors.initials: GA
authors.lastname: Petsko
authors.forename: T
authors.initials: T
authors.lastname: Oshima
mesh_terms: D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D007700:Kinetics; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology
chemical_list: D002413:Cations, Divalent; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase