Enzymatic characterization of a monomeric isocitrate dehydrogenase from Streptomyces lividans TK54 |
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| Authors: | Beibei Zhang Baojuan Wang Peng Wang Zhengyu Cao Enqi Huang Jiasheng Hao Antony M. Dean Guoping Zhu |
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| Affiliation: | 1. Key Laboratory of Molecular Evolution and Biodiversity and Institute of Molecular Biology and Biotechnology, College of Life Sciences, Anhui Normal University, Wuhu 241000, China;2. Key Laboratory of Biotic Environment and Ecological safety in Anhui Province, Anhui Normal University, Wuhu 241000, China;3. BioTechnology Institute and Department of Ecology, Evolution, and Behavior, University of Minnesota, St. Paul, MN 55108, USA;1. College of Environmental Science and Engineering, Hunan University, Changsha 410082, PR China;2. Key Laboratory of Environmental Biology and Pollution Control (Hunan University), Ministry of Education, Changsha 410082, PR China;3. College of Resources and Environment, Hunan Agricultural University, Changsha 410128, PR China;1. Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, Magdeburg D-39106, Germany;2. Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON, Canada M5S 3E5;3. Institute of Biomaterials and Biomedical Engineering, University of Toronto, 184 College Street, Toronto, ON, Canada, M5S3G9;1. Department of Microbial Engineering, College of Engineering, Konkuk University, Seoul 143-701, South Korea;2. Food Ingredients Center, Foods R&D, CheilJedang, Guro-dong, Guro-Gu, Seoul 152-051, South Korea;3. Center for Industrial Chemical Biotechnology, Ulsan Chemical R&BD Division, Korea Research Institute of Chemical Technology, P.O. Box 107, 141 Gajeong-ro, Yuseong-gu, Daejeon 305-600, South Korea;4. Department of Biological and Chemical Engineering, Hongik University, Sejong Ro 2639, Jochiwon, Sejong City 339-701, South Korea;5. Microbial Carbohydrate Resource Bank, Konkuk University, Seoul 143-701, South Korea;6. Institute for Ubiquitous Information Technology and Applications (CBRU), Konkuk University, Seoul 143-701, South Korea |
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| Abstract: | Isocitrate dehydrogenase (IDH) is one of the key enzymes in the citric acid cycle, which involves in providing energy and biosynthetic precursors for metabolism. Here, we report for the first time the enzymatic characterization of a monomeric NADP+-dependent IDH from Streptomyces lividans TK54 (SlIDH). The icd gene (GenBank database accession number EU661252) encoding IDH was cloned and overexpressed in Escherichia coli. The molecular mass of SlIDH was about 80 kDa, typical of a monomeric NADP-IDH, and showed high amino acid sequence identity with known monomeric IDHs. The optimal activity of the 6His-tagged SlIDH was found at pH values 8.5 (Mn2+) and 9.0 (Mg2+), and the optimal temperature was around 46 °C. Heat-inactivation studies showed that about 50% SlIDH activity was preserved at 38 °C after 20 min of incubation. The recombinant SlIDH displayed a 62,000-fold (kcat/Km) preference for NADP+ over NAD+ with Mn2+, and a 85,000-fold greater specificity for NADP+ than NAD+ with Mg2+. Therefore, SlIDH is a divalent cation-dependent monomeric IDH with remarkably high coenzyme preference for NADP+. |
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