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.
A comparative study of the possibilities of using ribokinase → phosphopentomutase → nucleoside phosphorylase cascades in the synthesis of modified nucleosides was carried out. Recombinant phosphopentomutase from Thermus thermophilus HB27 was obtained for the first time: a strain producing a soluble form of the enzyme was created, and a method for its isolation and chromatographic purification was developed. It was shown that cascade syntheses of modified nucleosides can be carried out both by the mesophilic and thermophilic routes from D-pentoses: ribose, 2-deoxyribose, arabinose, xylose, and 2-deoxy-2-fluoroarabinose. The efficiency of 2-chloradenine nucleoside synthesis decreases in the following order: Rib (92), dRib (74), Ara (66), F-Ara (8), and Xyl (2%) in 30 min for mesophilic enzymes. For thermophilic enzymes: Rib (76), dRib (62), Ara (32), F-Ara (<1), and Xyl (2%) in 30 min. Upon incubation of the reaction mixtures for a day, the amounts of 2-chloroadenine riboside (thermophilic cascade), 2-deoxyribosides (both cascades), and arabinoside (mesophilic cascade) decreased roughly by half. The conversion of the base to 2-fluoroarabinosides and xylosides continued to increase in both cases and reached 20-40%. Four nucleosides were quantitatively produced by a cascade of enzymes from D-ribose and D-arabinose. The ribosides of 8-azaguanine (thermophilic cascade) and allopurinol (mesophilic cascade) were synthesized. For the first time, D-arabinosides of 2-chloro-6-methoxypurine and 2-fluoro-6-methoxypurine were synthesized using the mesophilic cascade. Despite the relatively small difference in temperatures when performing the cascade reactions (50 and 80 °C), the rate of product formation in the reactions with Escherichia coli enzymes was significantly higher. E. coli enzymes also provided a higher content of the target products in the reaction mixture. Therefore, they are more appropriate for use in the polyenzymatic synthesis of modified nucleosides.
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