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°C the K(m) of E. coli IPMDH was 105 μM for IPM and 321 μM for NAD, the k(cat) was 69 s-1. The half denaturation temperature was 64°C, which was 20°C lower than that of the thermophile enzyme.