Abstract
3-Isopropylmalate dehydrogenase encoded by the Thiobacillus ferrooxidans leuB gene was purified to homogeneity from Escherichia coli cells harboring a recombinant plasmid containing the leuB gene. The native enzyme molecule is a dimer of molecular weight 38, 000. The Km value for 3-isopropylmalate was estimated to be 26 μM and that for NAD+ 0.8mM. The presence of K+ or NH4+ is essential for the enzyme reaction. The enzyme is activated about 4-fold by the addition of 1.0mM Mg2+ or Co2+. The optimum pH and temperature for the activity are 9.0 and 60°C, respectively. The properties of the enzyme are similar to those of the Salmonella typhimurium and Thermus thermophilus enzymes, except for substrate specificity. T. ferrooxidans 3-isopropylmalate dehydrogenase is able to utilize D- and L-malate as substrates in addition to 3-isopropylmalate. Sequencing of subcloned DNA revealed that the leuB gene consists of a 1, 074 by open reading frame and encodes 358 amino acid residues corresponding to the subunit (38, 462 Da). The amino acid sequence of 3-isopropylmalate dehydrogenase from T. ferrooxidans and those of some heterotrophic microorganisms have high homology.
