The Nature of Thermal Stability of Prokaryotic Nucleoside Phosphorylases

Abstract

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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ACKNOWLEDGMENTS

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).

Funding

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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The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.

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Translated by T. Tkacheva

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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). https://doi.org/10.1134/S0003683820060125

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Keywords:

  • nucleoside phosphorylase
  • site-directed mutagenesis
  • thermal stability