title: 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 ofD-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 fromThermus sp. 2.9, as well as two different genes of phosphoribosylpyrophosphate synthetase (PRPP-synthetase) and the adenine phosphoribosyltransferase (APR-transferase) gene fromThermus thermophilusHB27 into the expression vectors, generated high-yieldE. coliproducer strains, developed methods for the purification of the enzymes, and investigated enzyme substrate specificity. The enzymes were used for the conversion ofD-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 fromD-ribose and appropriate heterobases in one pot using a system of thermophilic enzymes in the presence of ATP, ribokinase, PRPP-synthetase, and APR-transferase.
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya Str., 16/10, Moscow, GSP-7, 117997, Russia.
authors.forename: R S
authors.initials: RS
authors.lastname: Esipov
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya Str., 16/10, Moscow, GSP-7, 117997, Russia.
authors.forename: Yu A
authors.initials: YA
authors.lastname: Abramchik
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya Str., 16/10, Moscow, GSP-7, 117997, Russia.
authors.forename: I V
authors.initials: IV
authors.lastname: Fateev
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya Str., 16/10, Moscow, GSP-7, 117997, Russia.
authors.forename: I D
authors.initials: ID
authors.lastname: Konstantinova
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya Str., 16/10, Moscow, GSP-7, 117997, Russia.
authors.forename: M A
authors.initials: MA
authors.lastname: Kostromina
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya Str., 16/10, Moscow, GSP-7, 117997, Russia.
authors.forename: T I
authors.initials: TI
authors.lastname: Muravyova
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya Str., 16/10, Moscow, GSP-7, 117997, Russia.
authors.forename: K G
authors.initials: KG
authors.lastname: Artemova
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya Str., 16/10, Moscow, GSP-7, 117997, Russia.
authors.forename: A I
authors.initials: AI
authors.lastname: Miroshnikov
keywords: adenine phosphoribosyltransferase; enzymatic nucleotide synthesis; phosphoribosylpyrophosphate synthetase; ribokinase; substrate properties; thermophilic microorganisms