Thermophilic phosphoribosyltransferases Thermus thermophilus HB27 in nucleotide synthesis.

Phosphoribosyltransferases are the tools that allow the synthesis of nucleotide analogues using multi-enzymatic cascades. The recombinant adenine phosphoribosyltransferase (TthAPRT) and hypoxanthine phosphoribosyltransferase (TthHPRT) from Thermus thermophilus HB27 were expressed in E. coli strains and purified by chromatographic methods with yields of 10-13 mg per liter of culture. The activity dependence of TthAPRT and TthHPRT on different factors was investigated along with the substrate specificity towards different heterocyclic bases. The kinetic parameters for TthHPRT with natural substrates were determined. Two nucleotides were synthesized: 9-(beta-D-ribofuranosyl)-2-chloroadenine 5'-monophosphate (2-Cl-AMP) using TthAPRT and 1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine-4-one 5'-monophosphate (Allop-MP) using TthHPRT.