Thermus thermophilus Nucleoside Phosphorylases Active in the Synthesis of Nucleoside Analogues
| Autor: | José V. Sinisterra, José Berenguer, Marcos Almendros |
|---|---|
| Přispěvatelé: | Comunidad de Madrid, Ministerio de Ciencia e Innovación (España), Fundación Ramón Areces |
| Rok vydání: | 2012 |
| Předmět: |
Purine
Pyrimidine Gene Expression Applied Microbiology and Biotechnology Substrate Specificity Nucleobase chemistry.chemical_compound Enzyme Stability Escherichia coli medicine Pentosyltransferases Cloning Molecular Enzymology and Protein Engineering Phosphorolysis chemistry.chemical_classification Ecology biology Nucleoside analogue Thermus thermophilus Temperature Nucleosides Hydrogen-Ion Concentration biology.organism_classification Recombinant Proteins Kinetics Enzyme Biochemistry chemistry Protein Multimerization Nucleoside Food Science Biotechnology medicine.drug |
| Zdroj: | Digital.CSIC. Repositorio Institucional del CSIC instname |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.07605-11 |
| Popis: | Cells extracts from Thermus thermophilus HB27 express phosphorolytic activities on purines and pyrimidine nucleosides. Five putative encoding genes were cloned and expressed in Escherichia coli, and the corresponding recombinant proteins were purified and studied. Two of these showed phosphorolytic activities against purine nucleosides, and third one showed phosphorolytic activity against pyrimidine nucleosides in vitro, and the three were named TtPNPI, TtPNPII, and TtPyNP, respectively. The optimal temperature for the activity of the three enzymes was beyond the water boiling point and could not be measured accurately, whereas all of them exhibited a wide plateau of optimal pHs that ranged from 5.0 to 7.0. Analytical ultracentrifugation experiments revealed that TtPNPI was a homohexamer, TtPNPII was a monomer, and TtPyNP was a homodimer. Kinetic constants were determined for the phosphorolysis of the natural substrates of each enzyme. Reaction tests with nucleoside analogues revealed critical positions in the nucleoside for its recognition. Activities with synthetic nucleobase analogues, such as 5-iodouracil or 2,6-diaminopurine, and arabinosides were detected, supporting that these enzymes could be applied for the synthesis of new nucleoside analogs with pharmacological activities. Comunidad Autónoma de Madrid; Ministry of Science and Innovation (BIO2010-18875, CTQ2009-11801); Fundación Ramón Areces |
| Databáze: | OpenAIRE |
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