Enzymatic production of non-natural nucleoside-5′-monophosphates by a thermostable uracil phosphoribosyltransferase

Abstract

The use of enzymes as biocatalysts applied to synthesis ofmodified nucleoside-5’-monophosphates (NMPs) is an interest-ing alternative to traditional multistep chemical methodswhich offers several advantages, such as stereo-, regio-, andenantioselectivity, simple downstream processing, and mild re-action conditions. Herein we report the recombinant expres-sion, production, and purification of uracil phosphoribosyl-transferase from Thermus themophilus HB8 (TtUPRT). The struc-ture of TtUPRT has been determined by protein crystallogra-phy, and its substrate specificity and biochemical characteris-tics have been analyzed, providing new structural insights intothe substrate-binding mode. Biochemical characterization ofthe recombinant protein indicates that the enzyme is a homo-tetramer, with activity and stability across a broad range oftemperatures (50–80 8C), pH (5.5–9) and ionic strength (0–500 mm NaCl). Surprisingly, TtUPRT is able to recognize several5 and 6-substituted pyrimidines as substrates. These experi-mental results suggest TtUPRT could be a valuable biocatalystfor the synthesis of modified NMPs.