3-Isopropylmalate dehydrogenase from chemolithoautotroph Thiobacillus ferrooxidans: DNA sequence, enzyme purification, and characterization.
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 microM 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 degrees 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.
Kawaguchi
H
H
Division of Bioresources Science, Faculty of Agriculture, Okayama University.
Inagaki
K
K
Kuwata
Y
Y
Tanaka
H
H
Tano
T
T
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate; D001483:Base Sequence; D011232:Chemical Precipitation; D002853:Chromatography, Liquid; D004269:DNA, Bacterial / Q000737:chemistry*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013855:Thiobacillus / Q000201:enzymology*
D000596:Amino Acids; D004269:DNA, Bacterial; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase; D000645:Ammonium Sulfate