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Heterologous Expression, Purification and Enzymatic Characterization of Isocitrate Dehydrogenases from Streptomyces Avermitilis MA-4680

Author: WangAo
Tutor: ZhuGuoPing
School: Anhui Normal University
Course: Biochemistry and Molecular Biology
Keywords: monomeric isocitrate dehydrogenase heterologous expression purification enzymatic characterization Streptomyces avermitilis MA-4680
CLC: Q814
Type: Master's thesis
Year: 2010
Downloads: 38
Quote: 0
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Abstract


Isocitrate dehydrogenase （IDH） belongs toβ-decarboxylating dehydrogenase family and catalyzes the oxidative decarboxylation from 2R, 3S-isocitrate to yield 2-oxoglutarate, CO2, and NAD（P）H, using NAD+ or NADP+ as coenzyme. It is one of the key enzymes in the TCA cycle, which involves in providing energy, biosynthetic precursors and reducing power for metabolism. NADP+-dependent IDH can be classified into monomeric and homodimeric structural forms. In the past decades, the dimeric NADP-IDHs （two subunits of 40-50 kDa each） have been extensively studied, including phylogeny, kinetics, catalytic and regulatory mechanism, and high-resolution crystal structures from numerous species. However, the information of monomeric NADP-IDHs is much less than that of dimeric NADP-IDHs.In this study, we reported the enzymatic characterization of a monomeric IDH from Streptomyces avermitilis MA-4680 （SaIDH）. The icd gene （GenBank database accession number NC003155） encoding IDH was cloned and successfully overexpressed in Escherichia coli. The molecular weight of SaIDH was about 80 kDa, typical of a monomeric NADP-IDH. SaIDH exhibited an absolute requirement for divalent cations, and Mn2+ was found to be the best metal ion activator. The optimal pH value of the 6His-tagged SaIDH was about pH 9.4 and the optimal temperature was around 45°C. Heat-inactivation studies showed that about 50% of SaIDH activity can be preserved at 37°C after incubation in water bath for 20 min in the presence of Mn2+. Although the purified SaIDH can function with both NAD+ and NADP+ in catalysis, it showed a 33,000-fold greater specificity for NADP+ than NAD+.This study provides the first experimental evidence that SaIDH is a divalent cation-dependent monomeric IDH which displays remarkably high coenzyme preference for NADP+. This work would provide a foundation for future investigation of the catalytic mechanism and tertiary structure of monomeric IDH and a basis for the research on the relationship of evolution between monomeric IDHs and homodimeic IDHs. Also, the information of SaIDH might be useful for metabolic engineering of secondary metabolite utilizations from S. avermitilis.

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