Applied Biochemistry and Biotechnology

, Volume 187, Issue 4, pp 1131–1142 | Cite as

Evaluation of the Potential Phosphorylation Effect on Isocitrate Dehydrogenases from Saccharomyces cerevisiae and Yarrowia lipolytica

  • Peng Wang
  • Tingting Liu
  • Xinxin Zhou
  • Guoping ZhuEmail author


Escherichia coli isocitrate dehydrogenase (IDH) is regulated by reversible phosphorylation on Ser113. Latest phosphoproteomic studies revealed that eukaryotic IDHs can also be phosphorylated on the analogous Ser site. So as to understand the possible phosphorylation mechanism, the equivalent Ser of NADP-IDHs from yeast Saccharomyces cerevisiae (ScIDH) and Yarrowia lipolytica(YlIDH) were investigated by site-directed mutagenesis. ScIDH Ser110 and YlIDH Ser103 were replaced by Asp or Glu to mimic a continuous phosphorylation state. Meanwhile, the effects of another four amino acids (Thr, Tyr, Gly, Ala) with various side chain on IDH activity were determined as well. Enzymatic analysis showed that replacement of Ser with Asp or Glu nearly inactivated ScIDH and YlIDH. Four other mutant enzymes of ScIDH, S110T, S110G, S110A, and S110Y, retained 38.07%, 3.24%, 2.65%, and 0.01% of its original activity, and four other mutant enzymes of YlIDH, S103T, S103G, S103A, and S103Y retained 44.26%, 27.99%, 16.29%, and 0.01% of its original activity, respectively. These results suggested that phosphorylation on eukaryotic IDHs has identical consequence to that on the bacterial IDHs. We thus presume that phosphorylation on the substrate-binding Ser shall be a common regulatory mechanism among IDHs.


Saccharomyces cerevisiae Yarrowia lipolytica Isocitrate dehydrogenase Phosphonationmechanism Site-directed mutagenesis 


Funding Information

This work was supported by the National Natural Science Foundation of China (31570010), the Provincial Project of Natural Science Research for Colleges and Universities of Anhui Province of China (KJ2018A0319) the Provincial Innovation Project for Oversea Talents in Anhui Province and Innovation Team of Scientific Research Platform in Anhui Universities.

Compliance with Ethical Standards

Conflicts of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Zhu, G., Golding, G. B., & Dean, A. M. (2005). The selective cause of an ancient adaptation. Science, 307(5713), 1279–1282.CrossRefGoogle Scholar
  2. 2.
    Ma, T., Peng, Y., Huang, W., & Ding, J. (2017). Molecular mechanism of the allosteric regulation of the αγ heterodimer of human NAD-dependent isocitrate dehydrogenase. Scientific Reports, 7(1), 40921.CrossRefGoogle Scholar
  3. 3.
    Ren, Z., Xiong, Y., Deng, C., & Jiang, S. (2012). Cloning, differential expression, and association analysis with fat traits of porcine IDH3γ gene. Applied Biochemistry and Biotechnology, 166(4), 1112–1120.CrossRefGoogle Scholar
  4. 4.
    Zhao, X. Y., Wang, P., Zhu, G. Y., Wang, B. J., & Zhu, G. P. (2014). Enzymatic characterization of a type II isocitrate dehydrogenase from pathogenic leptospira interrogans serovar Lai strain 56601. Applied Biochemistry and Biotechnology, 172(1), 487–496.CrossRefGoogle Scholar
  5. 5.
    Kim, H., Kim, S. H., Cha, H., Kim, S. R., Lee, J. H., & Park, J. W. (2016). IDH2 deficiency promotes mitochondrial dysfunction and dopaminergic neurotoxicity: implications for Parkinson’s disease. Free Radical Research, 50(8), 853–860.CrossRefGoogle Scholar
  6. 6.
    Thorsness, P. E., & Koshland, D. E., Jr. (1987). Inactivation of isocitrate dehydrogenase by phosphorylation is mediated by the negative charge of the phosphate. Journal of Biological Chemistry, 262(22), 10422–10425.Google Scholar
  7. 7.
    Dean, A. M., Lee, M. H., & Koshland, D. E., Jr. (1989). Phosphorylation inactivates Escherichia coli isocitrate dehydrogenase by preventing isocitrate binding. Journal of Biological Chemistry, 264(34), 20482–20486.Google Scholar
  8. 8.
    Hurley, J. H., Dean, A. M., Sohl, J. L., Koshland, D. E., Jr., & Stroud, R. M. (1990). Regulation of an enzyme by phosphorylation at the active site. Science, 249(4972), 1012–1016.CrossRefGoogle Scholar
  9. 9.
    Dean, A. M., & Koshland, D. E., Jr. (1990). Electrostatic and steric contributions to regulation at the active site of isocitrate dehydrogenase. Science, 249(4972), 1044–1046.CrossRefGoogle Scholar
  10. 10.
    LaPorte, D. C. (1993). The isocitrate dehydrogenase phosphorylation cycle: regulation and enzymology. Journal of Cellular Biochemistry, 51(1), 14–18.CrossRefGoogle Scholar
  11. 11.
    Singh, S. K., Matsuno, K., LaPorte, D. C., & Banaszak, L. J. (2001). Crystal structure ofbacillus subtilisisocitrate dehydrogenase at 1.55 Å. Journal of Biological Chemistry, 276(28), 26154–26163.CrossRefGoogle Scholar
  12. 12.
    Singh, S. K., Miller, S. P., Dean, A., Banaszak, L. J., & LaPorte, D. C. (2002). Bacillus subtilisisocitrate dehydrogenase. Journal of Biological Chemistry, 277(9), 7567–7573.CrossRefGoogle Scholar
  13. 13.
    Wang, P., Song, P., Jin, M., & Zhu, G. (2013). Isocitrate dehydrogenase from Streptococcus mutans: biochemical properties and evaluation of a putative phosphorylation Site at Ser102. PLoS One, 8(3), e58918.CrossRefGoogle Scholar
  14. 14.
    Ceccarelli, C., Grodsky, N. B., Ariyaratne, N., Colman, R. F., & Bahnson, B. J. (2002). Crystal structure of porcine mitochondrial NADP+-dependent isocitrate dehydrogenase complexed with Mn2+and isocitrate. Journal of Biological Chemistry, 277(45), 43454–43462.CrossRefGoogle Scholar
  15. 15.
    Xu, X., Zhao, J., Xu, Z., Peng, B., Huang, Q., Arnold, E., & Ding, J. (2004). Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity. Journal of Biological Chemistry, 279(32), 33946–33957.CrossRefGoogle Scholar
  16. 16.
    Peng, Y., Zhong, C., Huang, W., & Ding, J. (2008). Structural studies ofSaccharomyces cerevesiaemitochondrial NADP-dependent isocitrate dehydrogenase in different enzymatic states reveal substantial conformational changes during the catalytic reaction. Protein Science, 17(9), 1542–1554.CrossRefGoogle Scholar
  17. 17.
    Cozzone, A. J. (1998). Regulation of acetate metabolism by protein phosphorylation in enteric bacteria. Annual Review of Microbiology, 52(1), 127–164.CrossRefGoogle Scholar
  18. 18.
    Prasad, U. V., Vasu, D., Kumar, Y. N., Kumar, P. S., Yeswanth, S., Swarupa, V., Phaneendra, B. V., Chaudhary, A., & Sarma, P. V. G. K. (2013). Cloning, expression and characterization of NADP-dependent isocitrate dehydrogenase from Staphylococcus aureus. Applied Biochemistry and Biotechnology, 169(3), 862–869.CrossRefGoogle Scholar
  19. 19.
    Prasad, U. V., Vasu, D., Yeswanth, S., Swarupa, V., Sunitha, M. M., Choudhary, A., et al. (2015). Phosphorylation controls the functioning ofStaphylococcus aureusisocitrate dehydrogenase – favours biofilm formation. Journal of Enzyme Inhibition Medical Chemistry, 30(4), 655–661.CrossRefGoogle Scholar
  20. 20.
    Balganesh, T., Datta, S.&Ghosh, I. (2004). WIPO Patent Application WO/2004/ 087943 A1. 1–29.Google Scholar
  21. 21.
    Singh, V. K., & Ghosh, I. (2006). Kinetic modeling of tricarboxylic acid cycle and glyoxylate bypass in Mycobacterium tuberculosis, and its application to assessment of drug targets. Theoretical Biology and Medical Modelling, 3(1), 27.CrossRefGoogle Scholar
  22. 22.
    Mertins, P., Mani, D. R., Ruggles, K. V., Gillette, M. A., Clauser, K. R., Wang, P., et al. (2016). Proteogenomics connects somatic mutations to signalling in breast cancer. Nature, 534(7605), 55–62.CrossRefGoogle Scholar
  23. 23.
    Mertins, P., Yang, F., Liu, T., Mani, D. R., Petyuk, V. A., Gillette, M. A., Clauser, K. R., Qiao, J. W., Gritsenko, M. A., Moore, R. J., Levine, D. A., Townsend, R., Erdmann-Gilmore, P., Snider, J. E., Davies, S. R., Ruggles, K. V., Fenyo, D., Kitchens, R. T., Li, S., Olvera, N., Dao, F., Rodriguez, H., Chan, D. W., Liebler, D., White, F., Rodland, K. D., Mills, G. B., Smith, R. D., Paulovich, A. G., Ellis, M., & Carr, S. A. (2014). Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels. Molecular and Cellular Proteomics, 13(7), 1690–1704.CrossRefGoogle Scholar
  24. 24.
    Klammer, M., Kaminski, M., Zedler, A., Oppermann, F., Blencke, S., Marx, S., Müller, S., Tebbe, A., Godl, K., & Schaab, C. (2012). Phosphosignature predicts dasatinib response in non-small cell lung cancer. Molecular and Cellular Proteomics, 11(9), 651–668.CrossRefGoogle Scholar
  25. 25.
    Mertins, P., Qiao, J. W., Patel, J., Udeshi, N. D., Clauser, K. R., Mani, D. R., Burgess, M. W., Gillette, M. A., Jaffe, J. D., & Carr, S. A. (2013). Integrated proteomic analysis of post-translational modifications by serial enrichment. Nature Methods, 10(7), 634–637.CrossRefGoogle Scholar
  26. 26.
    Rigbolt, K. T., Prokhorova, T. A., Akimov, V., Henningsen, J., Johansen, P. T., Kratchmarova, I., et al. (2011). Science Signaling, 4, rs3.CrossRefGoogle Scholar
  27. 27.
    Lundby, A., Andersen, M. N., Steffensen, A. B., Horn, H., Kelstrup, C. D., Francavilla, C., et al. (2013). Science Signaling, 6, rs11.CrossRefGoogle Scholar
  28. 28.
    Li, X., Wang, P., Ge, Y., Wang, W., Abbas, A., & Zhu, G. P. (2013). NADP+-specific isocitrate dehydrogenase from oleaginous yeast yarrowia lipolytica CLIB122: biochemical characterization and coenzyme sites evaluation. Applied Biochemistry and Biotechnology, 171(2), 403–416.CrossRefGoogle Scholar
  29. 29.
    Pereira, J. M., Chevalier, C., Chaze, T., Gianetto, Q., Impens, F., Matondo, M., Cossart, P., & Hamon, M. A. (2018). Infection reveals a modification of SIRT2 critical for chromatin association. Cell Reports, 23(4), 1124–1137.CrossRefGoogle Scholar
  30. 30.
    Prezel, E., Elie, A., Delaroche, J., Stoppin-Mellet, V., Bosc, C., Serre, L., et al. (2017). Molecular Biology of the Cell, 29, 154–165.CrossRefGoogle Scholar
  31. 31.
    Boese, C. J., Nye, J., Buster, D. W., McLamarrah, T. A., Byrnes, A. E., Slep, K. C., Rusan, N. M., & Rogers, G. C. (2018). Asterless is a Polo-like kinase 4 substrate that both activates and inhibits kinase activity depending on its phosphorylation state. Molecular Biology of the Cell, 29(23), 2874–2886.CrossRefGoogle Scholar
  32. 32.
    Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., & Higgins, D. G. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23(21), 2947–2948.CrossRefGoogle Scholar
  33. 33.
    Gouet, P., Courcelle, E., Stuart, D. I., & Metoz, F. (1999). ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics, 15(4), 305–308.CrossRefGoogle Scholar
  34. 34.
    Wang, P., Lv, C., & Zhu, G. (2015). Novel type II and monomeric NAD+ specific isocitrate dehydrogenases: phylogenetic affinity, enzymatic characterization and evolutionary implication. Scientific Reports, 5(1), 9150.CrossRefGoogle Scholar
  35. 35.
    Zheng, J., & Jia, Z. (2010). Structure of the bifunctional isocitrate dehydrogenase kinase/phosphatase. Nature, 465(7300), 961–965.CrossRefGoogle Scholar
  36. 36.
    Karlstrom, M., Steen, I. H., Madern, D., Fedoy, A. E., Birkeland, N. K., & Ladenstein, R. (2006). The crystal structure of a hyperthermostable subfamily II isocitrate dehydrogenase from Thermotoga maritima. FEBS Journal, 273(13), 2851–2868.CrossRefGoogle Scholar
  37. 37.
    Leiros, H. K., Fedoy, A. E., Leiros, I., & Steen, I. H. (2012). The complex structures of isocitrate dehydrogenase fromClostridium thermocellumandDesulfotalea psychrophilasuggest a new active site locking mechanism. FEBS Open Bio, 2(1), 159–172.CrossRefGoogle Scholar
  38. 38.
    Matsuno, K., Blais, T., Serio, A. W., Conway, T., Henkin, T. M., & Sonenshein, A. L. (1999). Metabolic imbalance and sporulation in an isocitrate dehydrogenase mutant of Bacillus subtilis. Journal of Bacteriology, 181(11), 3382–3391.Google Scholar

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Authors and Affiliations

  1. 1.Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, College of Life SciencesAnhui Normal UniversityWuhuChina

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