Biochemical and molecular characterization of the NAD(+)-dependent isocitrate dehydrogenase from the chemolithotroph Acidithiobacillus thiooxidans.
An isocitrate dehydrogenase (ICDH) with an unique coenzyme specificity from Acidithiobacillus thiooxidans was purified and characterized, and its gene was cloned. The native enzyme was homodimeric with a subunit of M(r) 45000 and showed a 78-fold preference for NAD(+) over NADP(+). The cloned ICDH gene (icd) was expressed in an icd-deficient strain of Escherichia coli EB106; the activity was found in the cell extract. The gene encodes a 429-amino acid polypeptide and is located between open reading frames encoding a putative aconitase gene (upstream of icd) and a putative succinyl-CoA synthase beta-subunit gene (downstream of icd). A. thiooxidans ICDH showed high sequence similarity to bacterial NADP(+)-dependent ICDH rather than eukaryotic NAD(+)-dependent ICDH, but the NAD(+)-preference of the enzyme was suggested due to residues conserved in the coenzyme binding site of the NAD(+)-dependent decarboxylating dehydrogenase.
Inoue
Hiroyuki
H
Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, 1-1-1 Tsushima-naka, Japan.
Tamura
Takashi
T
Ehara
Nagisa
N
Nishito
Akira
A
Nakayama
Yumi
Y
Maekawa
Makiko
M
Imada
Katsumi
K
Tanaka
Hidehiko
H
Inagaki
Kenji
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000235:genetics; D007521:Isocitrate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D009243:NAD; D007521:Isocitrate Dehydrogenase