A Cascade of Thermophilic Enzymes As an Approach to the Synthesis of Modified Nucleotides
| Autor: | Esipov, R. S., Abramchik, Y. A., Fateev, I. V., Konstantinova, I. D., Maria Kostromina, Muravyova, T. I., Artemova, K. G., Miroshnikov, A. I. |
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| Předmět: |
adenine phosphoribosyltransferase
enzymatic nucleotide synthesis phosphoribosylpyrophosphate synthetase ENZYMATIC NUCLEOTIDE SYNTHESIS RIBOKINASE PHOSPHORIBOSYLPYROPHOSPHATE SYNTHETASE ADENINE PHOSPHORIBOSYLTRANSFERASE THERMOPHILIC MICROORGANISMS SUBSTRATE PROPERTIES ribokinase Molecular Biology thermophilic microorganisms substrate properties Research Article |
| Zdroj: | Scopus-Elsevier Acta Naturae ResearcherID |
| Popis: | We propose a new approach for the synthesis of biologically important nucleotides which includes a multi-enzymatic cascade conversion of D-pentoses into purine nucleotides. The approach exploits nucleic acid exchange enzymes from thermophilic microorganisms: ribokinase, phosphoribosylpyrophosphate synthetase, and adenine phosphoribosyltransferase. We cloned the ribokinase gene from Thermus sp. 2.9, as well as two different genes of phosphoribosylpyrophosphate synthetase (PRPP-synthetase) and the adenine phosphoribosyltransferase (APR-transferase) gene from Thermus thermophilus HB27 into the expression vectors, generated high-yield E. coli producer strains, developed methods for the purification of the enzymes, and investigated enzyme substrate specificity. The enzymes were used for the conversion of D-pentoses into 5-phosphates that were further converted into 5-phospho-α-D-pentofuranose 1-pyrophosphates by means of ribokinase and PRPP-synthetases. Target nucleotides were obtained through the condensation of the pyrophosphates with adenine and its derivatives in a reaction catalyzed by APR-transferase. 2-Chloro- and 2-fluoroadenosine monophosphates were synthesized from D-ribose and appropriate heterobases in one pot using a system of thermophilic enzymes in the presence of ATP, ribokinase, PRPP-synthetase, and APR-transferase. |
| Databáze: | OpenAIRE |
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