Deoxynucleoside Kinases Encoded by the yaaG andyaaF Genes of Bacillus subtilis

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 K m value of 6 M compared with 36 M for ATP. In addition, the K m for dGuo was 0.6 M with UTP but 6.5 M 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 M of dGuo, substrate inhibition was observed, but only with UTP as phosphate donor.

Structural Basis for the Preference of UTP over ATP in Human Deoxycytidine Kinase: Illuminating the Role of Main-Chain Reorganization

“…That dCK can utilize nucleoside triphosphates other than ATP is not surprising. In fact, most nucleoside and nucleoside monophosphate kinases can accept several nucleoside triphosphates as substrates (27). However, in most of these cases, ATP is assumed to be the preferred phosphoryl donor in the cellular environment.…”

“…The dNK is able to phosphorylate all four natural deoxynucleosides at a similar rate, though K m values vary by up to 3 orders of magnitude using ATP as phosphoryl donor (11). Like human dCK, dGK from Bacillus subtilis has been shown to be much more effective with UTP than ATP (27). The structure of human dGK unexpectedly contained the phosphoryl donor ATP in the acceptor binding site, while dNK was solved with dTTP, dThy, or dC in the acceptor binding site (25,26).…”

Functional Cloning, Heterologous Expression, and Purification of Two Different N-Deoxyribosyltransferases fromLactobacillus helveticus

“…These lactobacilli possess kinases for the four deoxyribonucleosides and in L. acidophilus R-26, three of the four activities are organized into two heterodimers deoxyadenosine/deoxycytidine kinase and deoxyadenosine/deoxyguanosine kinase (36). This latter enzyme could phosphorylate deoxyinosine to give dIMP, similar to the Bacillus subtilis deoxyguanosine kinase (37), which in turn would be converted to dITP through the successive action of guanylate kinase and nucleoside diphosphate kinase. Incorporation of dITP into DNA would be mutagenic and consequently detrimental for the cell.…”

The Role of Thymidine Kinases in the Activation of Pyrimidine Nucleoside Analogues

“…Most bacteria [17][18][19] and many DNA viruses (such as bacteriophages, vaccinia viruses, poxviruses and herpes viruses) code for proteins with thymidine kinase activity [1,2,9]. Interestingly, the Pox-virus TKs have similar characteristics as TK1, whereas the Herpes virus TKs have very broad specificity and they belong to the same enzyme family as dCK and TK2, as will be described below.…”