title: Heteroexpression and characterization of a monomeric isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680.
abstract: A monomeric NADP-dependent isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680 (SaIDH) was heteroexpressed in Escherichia coli, and the His-tagged enzyme was further purified to homogeneity. The molecular weight of SaIDH was about 80 kDa which is typical for monomeric isocitrate dehydrogenases. Structure-based sequence alignment reveals that the deduced amino acid sequence of SaIDH shows high sequence identity with known momomeric isocitrate dehydrogenase, and the coenzyme, substrate and metal ion binding sites are completely conserved. The optimal pH and temperature of SaIDH were found to be pH 9.4 and 45°C, respectively. Heat-inactivation studies showed that heating for 20 min at 50°C caused a 50% loss in enzymatic activity. In addition, SaIDH was absolutely specific for NADP+ as electron acceptor. Apparent Km values were 4.98 μM for NADP+ and 6,620 μM for NAD+, respectively, using Mn2+ as divalent cation. The enzyme performed a 33,000-fold greater specificity (kcat/Km) for NADP+ than NAD+. Moreover, SaIDH activity was entirely dependent on the presence of Mn2+ or Mg2+, but was strongly inhibited by Ca2+ and Zn2+. Taken together, our findings implicate the recombinant SaIDH is a divalent cation-dependent monomeric isocitrate dehydrogenase which presents a remarkably high cofactor preference for NADP+.
authors.affiliation: 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.
authors.forename: Ao
authors.initials: A
authors.lastname: Wang
authors.forename: Zheng-Yu
authors.initials: ZY
authors.lastname: Cao
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.forename: Ai-Min
authors.initials: AM
authors.lastname: Liu
authors.forename: Wei
authors.initials: W
authors.lastname: Pan
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.forename: Guo-Ping
authors.initials: GP
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007202:Indicators and Reagents / Q000494:pharmacology; D007477:Ions; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011387:Prokaryotic Cells / Q000187:drug effects / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000187:drug effects / Q000201:enzymology*; D013696:Temperature
chemical_list: D007202:Indicators and Reagents; D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D007521:Isocitrate Dehydrogenase