High yielding cascade enzymatic synthesis of 5-methyluridine using a novel Purine Nucleoside Phosphorylase, from Bacillus halodurans Alk36

Abstract

5-Methyluridine(5-MU) is a non-natural nucleoside that can be used as an intermediate in the synthesis of thymidine, and in the synthesis of nucleoside analogues AZT and stavudine, both of which are used in Highly Active Anti-Retroviral Treatment (HAART) of HIV/AIDS patients. 5-MU can be synthesised through the transglycosylation of D-ribose-1-phosphate, using guanosine as a donor, and thymine as receptor. However,the reagents guanosine and thymine are relatively insoluble, resulting in particulate substrates with poor reaction kinetics, and the most effective method of solubilising these materials is in hot aqueous solutions. It would therefore be preferable to utilize thermostable enzymes. The present work investigated the purine nucleoside phosphorylase (BHPNP1) present in the moderately thermophilic and alkaliphilic organism, Bacillus halodurans Alk361,2. The authors report on the combination of that enzyme with the uridine phosphorylase from E.coli in a one-pot cascade reaction to produce 5-methyl uridine in high yield.