Abstract
The overlapping yaaG and yaaF genes from Bacillus subtilis were 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 (Møllgaard, 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 Km value of 6 microm compared with 36 microm for ATP. In addition, the Km for dGuo was 0.6 microm with UTP but 6.5 microm 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 potent competitive inhibitor with respect to UTP. Above 30 microm of dGuo, substrate inhibition was observed, but only with UTP as phosphate donor.
Highlights
Biosynthesis of 2Ј-deoxyribosyl groups occurs solely through reduction of the 2Ј-hydroxyl group of ribonucleoside di- or triphosphates, catalyzed by ribonucleotide reductases [1, 2]
The present report concerns the molecular cloning of two overlapping B. subtilis genes encoding two homodimeric enzymes, deoxyguanosine kinase (dGK) and deoxyadenosine kinase (dAK)/deoxycytidine kinase (dCK), and describes the purification and characterization of the recombinant dGK overexpressed in E. coli
The genetic and enzymatic basis for the ability of B. subtilis to phosphorylate dCyd, dAdo, and dGuo was established by cloning and overexpressing the overlapping yaaG and yaaF genes in E. coli, an organism unable to phosphorylate deoxyribonucleosides other than dThd [16]
Summary
Biosynthesis of 2Ј-deoxyribosyl groups occurs solely through reduction of the 2Ј-hydroxyl group of ribonucleoside di- or triphosphates, catalyzed by ribonucleotide reductases [1, 2]. A deoxynucleoside kinase with a very different substrate specificity was recently characterized from Drosophila melanogaster. In this organism a single homodimeric enzyme (Dm-dNK) is capable of phosphorylating all four deoxyribonucleosides, with widely different efficiencies [11,12,13]. Among eubacteria only two genera, Lactobacilli [14] and Bacilli [15], have been shown to phosphorylate all four deoxynucleosides, whereas it has been established that a number of bacteria, including Escherichia coli and Salmonella enterica serovar Typhi, are lacking dGK, dAK, and dCK activities [16]. In Lactobacillus acidophilus strain R26, a strain that appears to lack a functional ribonucleotide reductase [14], the deoxynucleoside kinase activities are organized as three enzymes. The dAK/dCK enzyme appeared to be identical to the deoxyadenosine/deoxycytidine kinase previously purified and characterized from B. subtilis [15]
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