Author/Authors :
Esipov, R.S. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, russia , Kostromina, M.A. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, russia , Muravyova, T.I. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, russia , Artemova, K.G. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, russia , Miroshnikov, A.I. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, russia , Abramchik, Y.A. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, russia , Fateev I.V. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, russia , Konstantinova, I.D. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, russia
Abstract :
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 phosphoribo-syltransferase (APR-transferase) gene from Thermus thermophilus HB27 into expression vectors, generated high-yield E. coli producer strains, developed methods for the purification of the enzymes, and investigated en-zyme 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-syn-thetases. 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
Keywords :
enzymatic nucleotide synthesis , ribokinase , phosphoribosylpyrophosphate synthetase , adenine phos-phoribosyltransferase , thermophilic microorganisms , substrate properties
