TY - JOUR
T1 - 3-isopropylmalate dehydrogenase from chemolithoautotroph Thiobacillus ferrooxidans
T2 - DNA sequence, enzyme purification, and characterization
AU - Kawaguchi, Hiroshi
AU - Inagaki, Kenji
AU - Kuwata, Yasuyuki
AU - Tanaka, Hidehiko
AU - Tano, Tatsuo
PY - 1993/9
Y1 - 1993/9
N2 - 3-Isopropylmalate dehydrogenase encoded by the Thiobacillus ferrooxidans leuB gene was purified to homogeneity from Escherichia coli cells harboring a recombinant plasm id 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+ 0.8 mM. The presence of K+ or NH4+ is essential for the enzyme reaction. The enzyme is activated about 4-fold by the addition of 1.0 mM 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 bp 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.
AB - 3-Isopropylmalate dehydrogenase encoded by the Thiobacillus ferrooxidans leuB gene was purified to homogeneity from Escherichia coli cells harboring a recombinant plasm id 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+ 0.8 mM. The presence of K+ or NH4+ is essential for the enzyme reaction. The enzyme is activated about 4-fold by the addition of 1.0 mM 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 bp 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.
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U2 - 10.1093/oxfordjournals.jbchem.a124183
DO - 10.1093/oxfordjournals.jbchem.a124183
M3 - Article
C2 - 8282728
AN - SCOPUS:0027515241
VL - 114
SP - 370
EP - 377
JO - Journal of Biochemistry
JF - Journal of Biochemistry
SN - 0021-924X
IS - 3
ER -