The Nature of Thermal Stability of Prokaryotic Nucleoside Phosphorylases


A library of strains producing recombinant nucleoside phosphorylases (NPs) and their mutant and hybrid forms from various mesophilic and extremophilic microorganisms was constructed based on Escherichia coli cells. Substrates were shown to stabilize the NP structure upon thermal exposure, with the inorganic phosphate ion playing a decisive role in the process. Bioinformatics analyses made it possible to assume that the N‑terminal structure of NPs is largely responsible for their thermal stability. A hybrid thymidine phosphorylase (TPP) was constructed via the replacement of the N-terminal fragment (amino acid residues 1–62) of E. coli TPP with the corresponding TPP fragment from the thermophilic bacterium Geobacillus stearothermophilus. Higher thermal stability was observed for the hybrid TPP. The primary structure of E. coli uridine phosphorylase (UDP) was found to have a sequence, 25-Pro-Gly-Asp-Pro-30 (amino acid residues are numbered as in E. coli UDP), that is highly conserved among UDPs of mesophilic microorganisms. The E. coli UDP (Asp27Gly) mutant was constructed and similarly showed a higher thermal stability than the original form. The architecture of the phosphate-binding site and features of its function were assumed to be crucial for the thermal stability of the enzyme.

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  1. 1

    Liekens, S., De Clercq, E., and Neyts, J., Biochem. Pharmacol., 2001, vol. 61, no. 3, pp. 253–270.

    CAS  Article  Google Scholar 

  2. 2

    Carmeliet, P., Nature, 2005, vol. 438, no. 7070, pp. 932–936.

    CAS  Article  Google Scholar 

  3. 3

    Furukawa, T., Tabata, S., Yamamoto, M., Kawahara, K., Shinsato, Y., Minami, K., Shimokawa, M., and Akiyama, S., Pharmacol. Res., 2018, vol. 132, pp. 15–20.

  4. 4

    Bera, H. and Chigurupati, S., Eur. J. Med. Chem., 2016, vol. 124, pp. 992–1003.

    CAS  Article  Google Scholar 

  5. 5

    Wei Li and Hong Yue, Trend.Card. Med., 2018, vol. 28, no. 3, pp. 157–171.

    Google Scholar 

  6. 6

    Mikhailopulo, I.A. and Miroshnikov, A.I., Acta Naturae, 2010, vol. 2, no. 2, pp. 38–61.

    Article  Google Scholar 

  7. 7

    Kulikova, I.V., Drenichev, M.S., Solyev, P.N., Alexeev, C.S., and Mikhailov, S.N., Eur. J. Org. Chem., 2019, vol. 2019, no. 41, pp. 6999–7004.

    CAS  Article  Google Scholar 

  8. 8

    Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989.

    Google Scholar 

  9. 9

    Veiko, V.P., Siprashvili, Z.Z., Ratmanova, K.I., Gul’ko, L.B., Andryukhina, R.V., and Debabov, V.G., Biotekhnologoiya, 1994, no. 4, pp. 2–4.

  10. 10

    Veiko, V.P., Siprashvili, Z.Z., Ratmanova, K.I., Gul’ko, L.B., Mironov, A.A., Andryukhina, R.V., and Debabov, V.G., Dokl. Akad. Nauk SSSR, 1994, vol. 339, no. 6, pp. 819–821.

    CAS  Google Scholar 

  11. 11

    Mordkovich, N.N., Manuvera, V.A., Veiko, V.P., and Debabov, V.G., Biotekhnologiya, 2012, no. 1, pp. 21–30.

  12. 12

    Veiko, V.P., Chebotaev, D.A., Ovcharova, I.V., and Gul’ko, L.B., Russ. J. Bioorg. Chem., 1998, vol. 24, no. 5, pp. 335–340.

    Google Scholar 

  13. 13

    Antipov, A.N., Mordkovich, N.N., Khizhnyak, T.V., Okorokova, N.A., and Veiko, V.P., Appl. Biochem. Microbiol., 2020, vol. 56, no. 1, pp. 37–43.

    CAS  Article  Google Scholar 

  14. 14

    Veiko, V.P., Osipov, A.S., Shekhter, I.I., Bulenkov, M.T., Ratmanova, K.I., Gul’ko, L.B., Chibiskova, N.A., Errais, L.L., Derevshchikova, E.B., and Debabov, V.G., Bioorg. Khim., 1995, vol. 21, no. 5, pp. 354–358.

    CAS  PubMed  Google Scholar 

  15. 15

    Chebotaev, D.V., Gul’ko, L.B., and Veiko, V.P., Russ. J. Bioorg. Chem., 2001, vol. 27, no. 3, pp. 184–190.

    Article  Google Scholar 

  16. 16

    Bradford, M.M., Anal. Biochem., 1976, vol. 2, pp. 248–254.

    Article  Google Scholar 

  17. 17

    Laemmli, U.K., Nature, 1970, vol. 227, no. 5259, pp. 680–685.

    CAS  Article  Google Scholar 

  18. 18

    Leer, J.C., Hammer-Jespersen, K., and Schwartz, M., Eur. J. Biochem., 1977, vol. 75, no. 1, pp. 217–224.

    CAS  Article  Google Scholar 

  19. 19

    Cacciapuoti, G., Bertoldo, C., Brio, A., Zappia, V., and Porcelli, M., Extremophiles, 2003, vol. 7, pp. 159–168.

    CAS  Article  Google Scholar 

  20. 20

    Trivedi, S., Gehlot, H.S., and Rao, S.R.,Gen. Mol. Res., 2006, vol. 5, no. 4, pp. 816–827.

    CAS  Google Scholar 

  21. 21

    Gianese, G., Bossa, F., and Pascarella, S., Proteins, 2002, vol. 47, pp. 236–249.

    CAS  Article  Google Scholar 

  22. 22

    Vieille, C. and Zeikus, G.J., Microbiol. Mol. Biol. Rev., 2001, vol. 65, pp. 1–43.

    CAS  Article  Google Scholar 

  23. 23

    Brock, T.D., Science, 1967, vol. 158, pp. 1012–1019.

    CAS  Article  Google Scholar 

  24. 24

    Sawle, L. and Ghosh, K., Biophys. J., 2011, vol. 101, pp. 217–227.

    CAS  Article  Google Scholar 

  25. 25

    Kumar, S. and Nussinov, R., Cell. Mol. Life Sci, 2011, vol. 58, pp. 1216–1233.

  26. 26

    Karshikoff, A. and Ladenshtein, R., Prot. Eng., 1998, vol. 11, no. 10, pp. 867–872.

    CAS  Article  Google Scholar 

  27. 27

    Kamel, S., Thiele, I., Neubauer, P., and Wagner, S.S., Biochim. Biophys., Acta—Prot. Proteom., 2020, vol. 1868, no. 2.

  28. 28

    Visser, D.F., Hennessy, F., Rashamuse, J., Pletschke, B., and Brady, D., J. Mol. Cat. B: Enzym., 2011, vol. 68, pp. 279–285.

    CAS  Article  Google Scholar 

  29. 29

    Oliva, I., Zuffi, G., Barile, D., Orsini, G., Tonon, G., De Gioias, L., and Ghisotti, D., J. Biochem., 2004, vol. 135, no. 4, pp. 495–499.

    CAS  Article  Google Scholar 

  30. 30

    Luke, K.A., Higgins, C.L., and Wittung-Stafshede, P., FEBS J., 2007, vol. 274, pp. 4023–4033.

    CAS  Article  Google Scholar 

  31. 31

    Imanaka, T., Proc. Jpn. Acad., Ser., 2011, no. 9, pp. 587–602.

  32. 32

    Alemasov, N.A. and Fomin, E.S., Vavilov. Zh. Genet. Selekts., 2012, vol. 16, no. 4/1, pp. 774–783.

  33. 33

    Grishin, D.V., Pokrovskaya, M.V., Podobed, O.V., Gladilina, Yu.A., Pokrovskii, V.S., Aleksandrova, S.S., and Sokolov, N.N., Biomed. Khim., 2017, vol. 63, no. 2, pp. 124–131.

    CAS  Article  Google Scholar 

  34. 34

  35. 35

    Razvi, A. and Scholtz, J.M., Prot. Sci., 2006, vol. 15, no. 7, pp. 1569–1578.

    CAS  Article  Google Scholar 

  36. 36

    Mordkovich, N.N., Safonova, T.N., Antipov, A.N., Manuvera, V.A., Polyakov, K.M., Okorokova, N.A., and Veiko, V.P., Appl. Biochem. Microbiol., 2018, vol. 54, no. 1, pp. 16–25.

    Article  Google Scholar 

  37. 37

    Dumorne, K., Cordova, D.C., Astorga-Elo, M., and Renganathan, P., J. Microbiol. Biotechnol., 2017, vol. 27, no. 4, pp. 649–659.

    CAS  Article  Google Scholar 

  38. 38

    Pucci, F., Dhanani, M., Dehouck, Y., and Rooman, M., PLoS One, 2014, vol. 9, no. 3. e91659.

    Article  Google Scholar 

  39. 39

    Caradoc-Davies, T.T., Cutfield, S.M., Lamont, I.L., and Cutfield, J.F., J. Mol. Biol., 2004, vol. 337, no. 2, pp. 337–354.

    CAS  Article  Google Scholar 

  40. 40

    Pugmire, M.J. and Ealick, S.E., Structure, 1998, vol. 6, no. 11, pp. 1467–1479.

    CAS  Article  Google Scholar 

  41. 41

    Safonova, T.N., Mordkovich, N.N., Veiko, V.P., Okorokova, N.A., Manuvera, V.A., Dorovatovskii, P.V., Popov, V.O., and Polyakov, K.M., Acta Crystallogr. D: Struct. Biol., 2016, vol. 72, no. 2, pp. 203–210.

    CAS  Article  Google Scholar 

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This work was carried out on equipment of the Industrial Biotechnologies Collective Access Center of the Federal Research Center for Basics of Biotechnologies (Russian Academy of Sciences).


This work was supported by the Russian Foundation for Basic Research (project no. 18-04-00784A).

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Correspondence to V. P. Veiko.

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Mordkovich, N.N., Antipov, A.N., Okorokova, N.A. et al. The Nature of Thermal Stability of Prokaryotic Nucleoside Phosphorylases. Appl Biochem Microbiol 56, 662–670 (2020).

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  • nucleoside phosphorylase
  • site-directed mutagenesis
  • thermal stability