Dataset Information

The Peculiar Case of the Hyper-thermostable Pyrimidine Nucleoside Phosphorylase from Thermus thermophilus*.

ABSTRACT: The poor solubility of many nucleosides and nucleobases in aqueous solution demands harsh reaction conditions (base, heat, cosolvent) in nucleoside phosphorylase-catalyzed processes to facilitate substrate loading beyond the low millimolar range. This, in turn, requires enzymes that can withstand these conditions. Herein, we report that the pyrimidine nucleoside phosphorylase from Thermus thermophilus is active over an exceptionally broad pH (4-10), temperature (up to 100 °C) and cosolvent space (up to 80 % (v/v) nonaqueous medium), and displays tremendous stability under harsh reaction conditions with predicted total turnover numbers of more than 10⁶ for various pyrimidine nucleosides. However, its use as a biocatalyst for preparative applications is critically limited due to its inhibition by nucleobases at low concentrations, which is unprecedented among nonspecific pyrimidine nucleoside phosphorylases.

SUBMITTER: Kaspar F

PROVIDER: S-EPMC8247850 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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The Peculiar Case of the Hyper-thermostable Pyrimidine Nucleoside Phosphorylase from Thermus thermophilus*.

Kaspar Felix F Neubauer Peter P Kurreck Anke A

Chembiochem : a European journal of chemical biology 20210126 8

The poor solubility of many nucleosides and nucleobases in aqueous solution demands harsh reaction conditions (base, heat, cosolvent) in nucleoside phosphorylase-catalyzed processes to facilitate substrate loading beyond the low millimolar range. This, in turn, requires enzymes that can withstand these conditions. Herein, we report that the pyrimidine nucleoside phosphorylase from Thermus thermophilus is active over an exceptionally broad pH (4-10), temperature (up to 100 °C) and cosolvent space ...[more]

PMID: 33258231

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Project description:Nucleoside phosphorylases (NPs) are pivotal enzymes in the salvage pathway, catalyzing the reversible phosphorolysis of nucleosides to produce nucleobases and α-D-ribose 1-phosphate. Due to their efficiency in catalyzing nucleoside synthesis from purine or pyrimidine bases, these enzymes hold significant industrial importance in the production of nucleoside-based drugs. Given that the thermodynamic equilibrium for purine NPs (PNPs) is favorable for nucleoside synthesis-unlike pyrimidine NPs (PyNPs, UP, and TP)-multi-enzymatic systems combining PNPs with PyNPs, UPs, or TPs are commonly employed in the synthesis of nucleoside analogs. In this study, we report the first development of two engineered bifunctional fusion enzymes, created through the genetic fusion of purine nucleoside phosphorylase I (PNP I) and thymidine phosphorylase (TP) from Thermus thermophilus. These fusion constructs, PNP I/TP-His and TP/PNP I-His, provide an innovative one-pot, single-step alternative to traditional multi-enzymatic synthesis approaches. Interestingly, both fusion enzymes retain phosphorolytic activity for both purine and pyrimidine nucleosides, demonstrating significant activity at elevated temperatures (60-90 °C) and within a pH range of 6-8. Additionally, both enzymes exhibit high thermal stability, maintaining approximately 80-100% of their activity when incubated at 60-80 °C over extended periods. Furthermore, the transglycosylation capabilities of the fusion enzymes were explored, demonstrating successful catalysis between purine (2'-deoxy)ribonucleosides and pyrimidine bases, and vice versa. To optimize reaction conditions, the effects of pH and temperature on transglycosylation activity were systematically examined. Finally, as a proof of concept, these fusion enzymes were successfully employed in the synthesis of various purine and pyrimidine ribonucleoside and 2'-deoxyribonucleoside analogs, underscoring their potential as versatile biocatalysts in nucleoside-based drug synthesis.

Project description:BackgroundThermal stable α-glucosidases with transglycosylation activity could be applied to the industrial production of oligosaccharides as well as conjugation of sugars to biologically useful materials. Therefore, α-glucosidases isolated from thermophiles have gained attention over the past decade. In this study, the characterization of a highly thermostable α-glucosidase and its thermostability improved mutant from newly isolated strain Thermus thermophilus TC11 were investigated.ResultsThe recombinant α-glucosidase (TtAG) from Thermus thermophilus TC11 was expressed in Escherichia coli BL21 (DE3) and purified. The purified enzyme had a molecular mass of 184 kDa and consisted of 59-kDa subunits; it showed hydrolytic activity for pNP-α-D-glucopyranoside (pNPG), sucrose, trehalose, panose, and isomaltooligosaccharides and very low activity for maltose. The highest specific activity of 288.96 U/mg was observed for pNPG at 90 °C and pH 5.0; Pb(2+) provided a 20 % activity increase. TtAG was stable at 70 °C for more than 7 h and had a half-life of 195 min at 80 °C and 130 min at 90 °C. Transglycosylation activity was also observed with sucrose and trehalose as substrates. TtAG showed differences on substrate specificity, transglycosylation, multimerization, effects of metal ions and optimal pH from other reported Thermus α-glucosidases. One single-substitution TtAG mutant Q10Y with improved thermostability was also obtained from random mutagenesis library. The site-saturation mutagenesis and structural modelling analysis indicated that Q10Y substitution stabilized TtAG structure via additional hydrogen bonding and hydrophobic interactions.ConclusionOur findings indicate that TtAG is a highly thermostable and more acidic α-glucosidase distinct from other reported Thermus α-glucosidases. And this work also provides new insights into the catalytic and thermal tolerance mechanisms of α-glucosidases, which may guide molecular engineering of α-glucosidase and other thermostable enzymes for industrial application.

Dataset Information

The Peculiar Case of the Hyper-thermostable Pyrimidine Nucleoside Phosphorylase from Thermus thermophilus*.

Publications

The Peculiar Case of the Hyper-thermostable Pyrimidine Nucleoside Phosphorylase from Thermus thermophilus*.

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