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.
Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, 1-1-1 Tsushima-naka, Japan.
Hiroyuki
H
Inoue
Takashi
T
Tamura
Nagisa
N
Ehara
Akira
A
Nishito
Yumi
Y
Nakayama
Makiko
M
Maekawa
Katsumi
K
Imada
Hidehiko
H
Tanaka
Kenji
K
Inagaki
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