Review
Thermophilic nucleoside phosphorylases: Their properties, characteristics and applications

https://doi.org/10.1016/j.bbapap.2019.140304Get rights and content

Highlights

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    Nucleoside phosphorylases are valuable catalysts in the synthesis of nucleosides and their analogues.

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    Thermostable nucleoside phosphorylases withstand harsh reaction conditions such as high pH or the addition of organic solvents.

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    Thermostable nucleoside phosphorylases are stabilized by several mechanisms. Every thermophilic protein utilizes different mechanisms in a balanced way to achieve the required thermal stability.

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    Thermostable nucleoside phosphorylases have a wider substrate spectrum than their mesophilic counterparts.

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    Thermostable nucleoside phosphorylases are interesting biocatalysts for industrial applications.

Abstract

Nucleoside phosphorylases catalyze the reversible phosphorolysis of pyrimidine and purine nucleosides in the presence of phosphate. They are valuable catalysts in the synthesis of nucleosides and their analogues, which are often used as pharmaceuticals or their precursors. Thermostable nucleoside phosphorylases are promising biocatalysts, as they withstand harsh reaction conditions such as high pH or the addition of organic solvents. In this review, the characteristics and properties of thermostable nucleoside phosphorylases are described. Differences in amino acid content and protein structure were compared to their mesophilic homologues to identify features involved in thermostability. Substrate spectra of thermostable nucleoside phosphorylases were analyzed, and it is shown that thermostable nucleoside phosphorylases have a wider substrate spectrum than their mesophilic counterparts. Thus, thermostable nucleoside phosphorylases are interesting biocatalysts for industrial applications.

Introduction

Nucleoside phosphorylases (NPs) are enzymes belonging to the transferase family (EC 2.4) [1]. NPs catalyze the reversible phosphorolysis of nucleosides and their analogues to a nucleobase and an activated pentose moiety (α-D-pentofuranose-1-phosphate). While phosphorolysis reactions depend on inorganic phosphate, the absence of phosphate is favorable for synthesis reactions. NPs are classified into two families, NP-I and NP-II. NP-I family includes: purine nucleoside phosphorylase (PNP, EC 2.4.2.1), 5′-deoxy-5′-methylthioadenosine phosphorylase (MTAP, EC 2.4.2.28) and uridine phosphorylase (UP, EC 2.4.2.3). NP-II family includes thymidine phosphorylase (TP, EC 2.4.2.4) and pyrimidine nucleoside phosphorylase (PyNP, EC 2.4.2.2). The families differ mainly in the quaternary structures of the proteins, as well as in the active sites and favored substrates [[1], [2], [3]].

NPs from microorganisms were initially identified and characterized in mesophilic organisms as Escherichia coli (Ec) [4], Salmonella typhimurium [5], Bacillus subtilis (Bs) [6], Mycoplasma hyorhinis [7] and others. However, early on the use of mesophilic enzymes were found to be challenging for industrial applications [8]. This is mainly because mesophilic enzymes have a shorter half-life, especially under harsh reaction conditions (e.g. low or high temperatures or pH, addition of organic solvents). Indeed, Saunders et al. [9], showed that the crude extract of PyNP from Geobacillus stearothermophilus (GsPyNP) is more stable at higher temperatures compared to its equivalents from E. coli and B. subtilis. Extreme conditions are called for in some reactions to increase the solubility of the nucleoside or the nucleobase. Moreover, purification of recombinant thermophilic enzymes is less laborious since in many cases, particularly for hyperthermophilic biocatalysts, heat treatment leads to efficient removal of host proteins. Additionally, it was shown that mesophilic biocatalysts have a limited substrate spectrum [10,11]. These drawbacks led to the study of NPs derived from extremophilic organisms.

Extremophiles are microorganisms (archaea and bacteria) living in environments with extreme conditions like high temperature, high pressure, high salt concentrations and/or heavy metals [12]. Enzymes from such sources are called extremozymes and are able to catalyze reactions at different harsh conditions such as high temperatures, extreme pH (high or low), and/or in the presence of organic solvents. Thus, enzymes from these organisms are investigated as useful biocatalysts [8,13]. The focus has been on thermophilic enzymes that are stable at high temperatures ranging between 45 °C to 100 °C. Thermophiles have been grouped based on their optimal growth temperature; moderate thermophiles (45 °C - 70 °C), extreme thermophiles (> 70 °C) and hyperthermophiles (80 °C – 100 °C) [14].

Section snippets

Thermophilic nucleoside phosphorylases

PNPs are the most studied nucleoside phosphorylases due to their importance as drug targets and their application in the enzymatic synthesis of purine nucleoside analogues [3,15]. Nine thermophilic PNPs from seven thermophilic archaea and bacteria have been reported [10,[16], [17], [18], [19], [20], [21]] (Table 1). Thermus thermophilus (Tt) [18] and Geobacillus stearothermophilus (Gs) [20,21] have been reported to have two PNPs, denoted PNP I and PNP II. Only one UP, few MTAPs and PyNPs from

Protein sequences of thermostable NPs

The thermal stability of enzymes is thought to be due to several factors, including a large number of hydrogen bonds and/or salt bridges, increased compactness or hydrophobicity [[30], [31], [32]]. Salt bridges arise from the non-covalent interaction between negatively charged carboxylate ion (RCOO−) of glutamate (Glu) or aspartic acid (Asp) and the positively charged ammonium or guanidium groups (RNH3+ or RNHC(NH2)2+ groups) of lysine (Lys), arginine (Arg) or histidine (His), respectively.

Substrate spectrum of thermostable nucleoside phosphorylases

Substrate spectra of NPs follow general guidelines with few exceptions [1,3]. NP-I members are segregated into two categories, trimeric and hexameric proteins. Trimeric proteins include PNPs of mammalian origin and MTAPs. PNPs of mammalian origins are specific to C6-oxo-purines, whereas MTAPs accept both C6-oxo- and C6-amino-purines. Hexameric proteins encompass PNPs of bacterial origin which accepts both C6-oxo- and C6-amino- purines and UPs which are specific for C4-oxo- and

Synthesis of nucleoside analogues by thermostable NPs

For the synthesis of nucleoside analogues, NPs were employed either as free or immobilized enzymes (Table 3). Immobilization is a powerful tool for continuous processes and allows consecutive batch reactions with a higher overall yield [1]. Synthesized nucleoside analogues had modifications on the base moiety, sugar moiety or both. For the base modified nucleosides, the obtained yields were between 40% - 90%. However, fluoro modifications on the sugar moiety had a big impact on the nucleoside

Conclusion

Thermophilic nucleoside phosphorylases are valuable tools for the synthesis of clinically and biotechnologically relevant nucleoside analogues. Since operating at high temperatures improves the synthesis process, factors enhancing enzymes thermostability were evaluated for the reported thermophilic nucleoside phosphorylases. Our analysis suggests that every enzyme has its own unique way to achieve the required stability, usually by combining two or more factors. Some enzymes rely on salt

Declaration of Competing Interest

A.W. is CEO and P.N. a shareholder of the biotech startup BioNukleo GmbH. No conflict of interest is known for the other authors.

Acknowledgments

S.K. was funded by the Schlumberger foundation. The authors are thankful to Erik Wade for proofreading the manuscript and critical comments.

References (51)

  • S. Kumar et al.

    Relationship between ion pair geometries and electrostatic strengths in proteins

    Biophys. J.

    (2002)
  • M.L. Bagarolo et al.

    Multiple disulfide bridges modulate conformational stability and flexibility in hyperthermophilic archaeal purine nucleoside phosphorylase

    Biochim. Biophys. Acta

    (2015)
  • X. Zhou et al.

    Synthesis of 2,6-dihalogenated purine nucleosides by thermostable nucleoside phosphorylases

    Adv. Synth. Catal.

    (2015)
  • S. Zhu et al.

    Two-step efficient synthesis of 5-methyluridine via two thermostable nucleoside phosphorylase from Aeropyrum pernix

    Bioorganic. Med. Chem. Lett.

    (2012)
  • N. Hori et al.

    Production of 5-methyluridine by immobilized thermostable purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus JTS 859

    J. Biotechnol.

    (1991)
  • S.A. Ingale et al.

    7-Deaza-2′-deoxyguanosine: selective nucleobase halogenation, positional impact of space-occupying substituents, and stability of DNA with parallel and antiparallel strand orientation

    J. Org. Chem.

    (2016)
  • C.R. Chen et al.

    ProteinVolume: calculating molecular van der Waals and void volumes in proteins

    BMC Bioinformatics.

    (2015)
  • S. Kamel et al.

    Enzymatic synthesis of nucleoside analogues by nucleoside phosphorylases

  • M.J. Pugmire et al.

    Structural analyses reveal two distinct families of nucleoside phosphorylases

    Biochem. J.

    (2002)
  • H. Yehia et al.

    Substrate spectra of nucleoside phosphorylases and their potential in the production of pharmaceutically active compounds

    Curr. Pharm. Des.

    (2017)
  • N.G.G. Panova et al.

    Substrate specificity of Escherichia coli thymidine phosphorylase

    Biochem

    (2007)
  • J. Vande Voorde et al.

    Characterization of pyrimidine nucleoside phosphorylase of mycoplasma hyorhinis: implications for the clinical efficacy of nucleoside analogues

    Biochem. J.

    (2012)
  • J. Del Arco et al.

    Purine and pyrimidine salvage pathway in thermophiles: a valuable source of biocatalysts for the industrial production of nucleic acid derivatives

    Appl. Microbiol. Biotechnol.

    (2018)
  • X. Zhou et al.

    Recombinant purine nucleoside phosphorylases from thermophiles: preparation, properties and activity towards purine and pyrimidine nucleosides

    FEBS J.

    (2013)
  • K. Dumorné et al.

    Extremozymes: a potential source for industrial applications

    J. Microbiol. Biotechnol.

    (2017)

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