Deoxynucleoside kinases encoded by the yaaG and yaaF genes of Bacillus subtilis -: Substrate specificity and kinetic analysis of deoxyguanosine kinase with UTP as the preferred phosphate donor

The overlapping yaaG and yaaF genes from Bacillus subtilis mere cloned and overexpressed in Escherichia coli. Purification of the gene products showed that yaaG encoded a homodimeric deoxyguanosine kinase (dGK) and that yaaF encoded a homodimeric deoxynucleoside kinase capable of phosphorylating both deoxyadenosine and deoxycytidine. The latter was identical to a previously characterized dAdo/dCyd kinase (Mollgaard, H. (1980) J. Biol. Chem. 255, 8216-8220). The purified recombinant dGK( was highly specific toward 6-oxopurine 2'-deoxyribonucleosides as phosphate acceptors showing only marginal activities with Guo, dAdo, and 2',3'-dideoxyguanosine. UTP was the preferred phosphate donor with a K-m value of 6 muM compared with 36 muM for ATP. In addition, the K-m for dGuo was 0.6 muM with UTP but 6.5 muM with ATP as phosphate donor. The combination of these two effects makes UTP over 50 times more efficient than ATP. Initial velocity and product inhibition studies indicated that the reaction with dGuo and UTP as substrates followed an Ordered Bi Bi reaction mechanism with UTP as the leading substrate and UDP the last product to leave. dGTP was a patent competitive inhibitor with respect to UTP. Above 30 muM of dGuo, substrate inhibition was observed, but only with UTP as phosphate donor.