Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 370-377 |
| Number of pages | 8 |
| Journal | Journal of Biochemistry |
| Volume | 114 |
| Issue number | 3 |
| Publication status | Published - Sep 1993 |
Fingerprint
ASJC Scopus subject areas
- Statistics, Probability and Uncertainty
- Applied Mathematics
- Physiology (medical)
- Radiology Nuclear Medicine and imaging
- Molecular Biology
- Biochemistry
Cite this
3-isopropylmalate dehydrogenase from chemolithoautotroph Thiobacillus ferrooxidans : DNA sequence, enzyme purification, and characterization. / Kawaguchi, Hiroshi; Inagaki, Kenji; Kuwata, Yasuyuki; Tanaka, Hidehiko; Tano, Tatsuo.
In: Journal of Biochemistry, Vol. 114, No. 3, 09.1993, p. 370-377.Research output: Contribution to journal › Article
}
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.
UR - http://www.scopus.com/inward/record.url?scp=0027515241&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0027515241&partnerID=8YFLogxK
M3 - Article
VL - 114
SP - 370
EP - 377
JO - Journal of Biochemistry
JF - Journal of Biochemistry
SN - 0021-924X
IS - 3
ER -