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Heteroexpression and characterization of a monomeric isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680

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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 K m 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 (k cat/K m) 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+.

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Acknowledgments

This research was supported by funds from the National Natural Science Foundation of China (30500300; 30870062; 31040003), New Century Excellent Talents in University of the Education Ministry of China (NCET-06-0558), Scientific Research Foundation for the Returned Overseas Chinese Scholars from State Education Ministry, the Key Laboratory of Biotic Environment and Ecological Safety in Anhui Province and Program for Innovative Research Team in Anhui Normal University.

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  1. 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

    Ao Wang, Zheng-Yu Cao, Peng Wang, Ai-Min Liu, Wei Pan, Jie Wang & Guo-Ping Zhu

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  1. Ao Wang
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Correspondence to Guo-Ping Zhu.

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Wang, A., Cao, ZY., Wang, P. et al. Heteroexpression and characterization of a monomeric isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680. Mol Biol Rep 38, 3717–3724 (2011). https://doi.org/10.1007/s11033-010-0486-3

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