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Int. J. Mol. Sci. 2016, 17(3), 296; doi:10.3390/ijms17030296

Biochemical Characterization and Complete Conversion of Coenzyme Specificity of Isocitrate Dehydrogenase from Bifidobacterium longum

The Research Center of Life Omics and Health and Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu 241000, Anhui, China
These authors contributed equally to this work.
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Academic Editor: Ritva Tikkanen
Received: 10 January 2016 / Revised: 9 February 2016 / Accepted: 19 February 2016 / Published: 26 February 2016
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Abstract

Bifidobacterium longum is a very important gram-positive non-pathogenic bacterium in the human gastrointestinal tract for keeping the digestive and immune system healthy. Isocitrate dehydrogenase (IDH) from B. longum (BlIDH), a novel member in Type II subfamily, was overexpressed, purified and biochemically characterized in detail. The active form of BlIDH was an 83-kDa homodimer. Kinetic analysis showed BlIDH was a NADP+-dependent IDH (NADP-IDH), with a 567- and 193-fold preference for NADP+ over NAD+ in the presence of Mg2+ and Mn2+, respectively. The maximal activity for BlIDH occurred at 60 °C (with Mn2+) and 65 °C (with Mg2+), and pH 7.5 (with Mn2+) and pH 8.0 (with Mg2+). Heat-inactivation profiles revealed that BlIDH retained 50% of maximal activity after incubation at 45 °C for 20 min with either Mn2+ or Mg2+. Furthermore, the coenzyme specificity of BlIDH can be completely reversed from NADP+ to NAD+ by a factor of 2387 by replacing six residues. This current work, the first report on the coenzyme specificity conversion of Type II NADP-IDHs, would provide better insight into the evolution of NADP+ use by the IDH family. View Full-Text
Keywords: Bifidobacterium longum; isocitrate dehydrogenase; biochemical characterization; coenzyme specificity determinants; kinetics Bifidobacterium longum; isocitrate dehydrogenase; biochemical characterization; coenzyme specificity determinants; kinetics
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Huang, S.-P.; Cheng, H.-M.; Wang, P.; Zhu, G.-P. Biochemical Characterization and Complete Conversion of Coenzyme Specificity of Isocitrate Dehydrogenase from Bifidobacterium longum. Int. J. Mol. Sci. 2016, 17, 296.

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