3-isopropylmalate dehydrogenase from chemolithoautotroph Thiobacillus ferrooxidans

DNA sequence, enzyme purification, and characterization

Hiroshi Kawaguchi, Kenji Inagaki, Yasuyuki Kuwata, Hidehiko Tanaka, Tatsuo Tano

Research output: Contribution to journalArticle

20 Citations (Scopus)

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 languageEnglish
Pages (from-to)370-377
Number of pages8
JournalJournal of Biochemistry
Volume114
Issue number3
Publication statusPublished - Sep 1993

Fingerprint

3-Isopropylmalate Dehydrogenase
Thiobacillus
DNA sequences
Purification
DNA Sequence
Enzymes
Genes
Gene
Amino acids
Substrate
Thermus thermophilus
Amino Acids
Salmonella
Microorganisms
Dimer
Substrates
Salmonella typhimurium
Amino Acid Sequence
Substrate Specificity
DNA Sequence Analysis

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 journalArticle

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