PURIFICATION, CATALYTIC PROPERTIES AND THERMOSTABILITY OF 3-ISOPROPYLMALATE DEHYDROGENASE FROM ESCHERICHIA-COLI

3-isopropylmalate dehydrogenase (IPMDH) from Escherichia coli was over expressed, purified and crystallized. The enzyme was characterized and compared to its thermophilic counterpart from Thermus thermophilus st rain HB8. As in the thermophile enzyme, the activity of E. coli IPMDH was dependent on the divalent cations, Mg2+ or Mn2+, with Mn2+ being t he 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 mu M for IPM and 321 mu M for NAD, the k(cat) was 69 s(-1). The half denaturation temperature was 64 degrees C, whi ch was 20 degrees C lower than that of the thermophile enzyme.