A Cascade of Thermophilic Enzymes As an Approach to the Synthesis of Modified Nucleotides

Abstract

We propose a new approach for the synthesis of biologically important nucleotides which includes a multi-enzymatic cascade conversion of <i>D</i>-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 <i>Thermus sp</i>. 2.9, as well as two different genes of phosphoribosylpyrophosphate synthetase (PRPP-synthetase) and the adenine phosphoribosyltransferase (APR-transferase) gene from <i>Thermus thermophilus</i> HB27 into the expression vectors, generated high-yield <i>E. coli</i> producer strains, developed methods for the purification of the enzymes, and investigated enzyme substrate specificity. The enzymes were used for the conversion of <i>D</i>-pentoses into 5-phosphates that were further converted into 5-phospho-α-<i>D</i>-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 <i>D</i>-ribose and appropriate heterobases in one pot using a system of thermophilic enzymes in the presence of ATP, ribokinase, PRPP-synthetase, and APR-transferase.