Expression and characterization of a novel isocitrate dehydrogenase from Streptomyces diastaticus No. 7 strain M1033.
Isocitrate dehydrogenase (IDH) is one of the key enzymes in tricarboxylic acid cycle, widely distributed in Archaea, Bacteria and Eukarya. Here, we report for the first time the cloning, expression and characterization of a monomeric NADP(+)-dependent IDH from Streptomyces diastaticus No. 7 strain M1033 (SdIDH). Molecular mass of SdIDH was about 80 kDa and showed high amino acid sequence identity with known monomeric IDHs. Maximal activity of SdIDH was observed at pH 8.0 (Mn(2+)) and 9.0 (Mg(2+)), and the optimal temperature was 40 °C (Mn(2+)) and 37 °C (Mg(2+)). Heat-inactivation studies showed that SdIDH remained about 50 % activity after 20 min of incubation at 47 °C. SdIDH displayed a 19,000 and 32,000-fold (k                 (cat)/K                 (m)) preference for NADP(+) over NAD(+) with Mn(2+) and Mg(2+), respectively. Our work implicate that SdIDH is a divalent metal ion-dependent monomeric IDH with remarkably high coenzyme preference for NADP(+). This work may provide fundamental information for further investigation on the catalytic mechanism of monomeric IDH and give a clue to disclose the real cause of IDH monomerization.
Key Laboratory of Molecular Evolution and Biodiversity and Institute of Molecular Biology and Biotechnology, College of Life Sciences, Anhui Normal University, No.1 Beijing East Road, Wuhu, 241000, Anhui, People's Republic of China.
Bei-Bei
BB
Zhang
Peng
P
Wang
Ao
A
Wang
Wen-Cai
WC
Wang
Wang-Gang
WG
Tang
Guo-Ping
GP
Zhu
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004795:Enzyme Stability; D004926:Escherichia coli; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology*
D001426:Bacterial Proteins; D008345:Manganese; D007521:Isocitrate Dehydrogenase