Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
ReviewThermophilic nucleoside phosphorylases: Their properties, characteristics and 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.
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