Enzymatic characterization of a monomeric isocitrate dehydrogenase from Streptomyces lividans TK54

Beibei Zhang; Baojuan Wang; Peng Wang; Zhengyu Cao; Enqi Huang; Jiasheng Hao; Antony M. Dean; Guoping Zhu

doi:10.1016/j.biochi.2009.07.011

Enzymatic characterization of a monomeric isocitrate dehydrogenase from Streptomyces lividans TK54

Beibei Zhang, Baojuan Wang, Peng Wang, Zhengyu Cao, Enqi Huang, Jiasheng Hao, Antony M. Dean, Guoping Zhu

Biotechnology Institute

Research output: Contribution to journal › Article › peer-review

22 Scopus citations

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 (k_cat/K_m) 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⁺.

Original language	English (US)
Pages (from-to)	1405-1410
Number of pages	6
Journal	Biochimie
Volume	91
Issue number	11-12
DOIs	https://doi.org/10.1016/j.biochi.2009.07.011
State	Published - Nov 2009

Bibliographical note

Funding Information:
We are grateful to Professor Congzhao Zhou and his laboratory for their kind assistances. This research was supported by funds from the National Natural Science Foundation of China (30500300; 30870062), New Century Excellent Talents in University of the Education Ministry of China (NCET-06-0558), the Outstanding Youth Foundation of Science and Technology of Anhui Province (06043089; 08040106811) and the Key Grant of Anhui Bureau of Education (2006KJ061A).

Keywords

Coenzyme specificity
Kinetics
Monomeric isocitrate dehydrogenase
Streptomyces lividans
Western blot

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title = "Enzymatic characterization of a monomeric isocitrate dehydrogenase from Streptomyces lividans TK54",

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+.",

keywords = "Coenzyme specificity, Kinetics, Monomeric isocitrate dehydrogenase, Streptomyces lividans, Western blot",

author = "Beibei Zhang and Baojuan Wang and Peng Wang and Zhengyu Cao and Enqi Huang and Jiasheng Hao and Dean, {Antony M.} and Guoping Zhu",

note = "Funding Information: We are grateful to Professor Congzhao Zhou and his laboratory for their kind assistances. This research was supported by funds from the National Natural Science Foundation of China (30500300; 30870062), New Century Excellent Talents in University of the Education Ministry of China (NCET-06-0558), the Outstanding Youth Foundation of Science and Technology of Anhui Province (06043089; 08040106811) and the Key Grant of Anhui Bureau of Education (2006KJ061A).",

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AU - Hao, Jiasheng

AU - Dean, Antony M.

AU - Zhu, Guoping

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Enzymatic characterization of a monomeric isocitrate dehydrogenase from Streptomyces lividans TK54

Abstract

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Keywords

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