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NADP+-Specific Isocitrate Dehydrogenase from Oleaginous Yeast Yarrowia lipolytica CLIB122: Biochemical Characterization and Coenzyme Sites Evaluation

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Abstract

NADP+-dependent isocitrate dehydrogenase from Yarrowia lipolytica CLIB122 (YlIDP) was overexpressed and purified. The molecular mass of YlIDP was estimated to be about 81.3 kDa, suggesting its homodimeric structure in solution. YlIDP was divalent cation dependent and Mg2+ was found to be the most favorable cofactor. The purified recombinant YlIDP displayed maximal activity at 55 °C and its optimal pH for catalysis was found to be around 8.5. Heat inactivation studies revealed that the recombinant YlIDP was stable below 45 °C, but its activity dropped quickly above this temperature. YlIDP was absolutely dependent on NADP+ and no NAD-dependent activity could be detected. The K m values displayed for NADP+ and isocitrate were 59 and 31 μM (Mg2+), 120 μM and 58 μM (Mn2+), respectively. Mutant enzymes were constructed to tentatively alter the coenzyme specificity of YlIDP. The K m values for NADP+ of R322D mutant was 2,410 μM, being about 41-fold higher than that of wild type enzyme. NAD+-dependent activity was detected for R322D mutant and the K m and k cat values for NAD+ were 47,000 μM and 0.38 s−1, respectively. Although the R322D mutant showed low activity with NAD+, it revealed the feasibility of engineering an eukaryotic IDP to a NAD+-dependent one.

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Acknowledgments

This research was supported by funds from the National High Technology Research and Development Program (“863” Program: 2012AA02A708), the National Natural Science Foundation of China (31170005 and 30870062), Specialized Research Fund for the Doctoral Program of Higher Education of China (20113424110004), the Fund of State Key Laboratory of Genetics Resources and Evolution from Kunming Institute of Zoology (Chinese Academy of Sciences, CAS) (GREKF11-07), and Anhui Provincial Natural Science Foundation (1208085QC52 and 1308085QC67).

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Authors and Affiliations

  1. Institute of Molecular Biology and Biotechnology and Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu, 241000, Anhui, China

    Xue Li, Peng Wang, Yadong Ge & Guoping Zhu

  2. State Key Laboratory of Genetics Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223, Yunnan, China

    Wen Wang

  3. College of Life Science and Technology, Xinjiang University, Urumqi, 830046, Xinjiang, China

    Abdulla Abbas

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  1. Xue Li
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  2. Peng Wang
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  3. Yadong Ge
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  4. Wen Wang
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  5. Abdulla Abbas
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  6. Guoping Zhu
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Correspondence to Guoping Zhu.

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Li, X., Wang, P., Ge, Y. et al. NADP+-Specific Isocitrate Dehydrogenase from Oleaginous Yeast Yarrowia lipolytica CLIB122: Biochemical Characterization and Coenzyme Sites Evaluation. Appl Biochem Biotechnol 171, 403–416 (2013). https://doi.org/10.1007/s12010-013-0373-1

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