Identification of residues involved in the specificity and regulation of the highly efficient multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster
W. Knecht et al., Identification of residues involved in the specificity and regulation of the highly efficient multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster, J MOL BIOL, 301(4), 2000, pp. 827-837
In contrast to all known deoxyribonucleoside kinases, a single highly effic
ient deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) is ab
le to phosphorylate all precursor nucleosides for DNA synthesis. Dm-dNK was
mutated in vitro by high-frequency random mutagenesis, expressed in the th
ymidine kinase-deficient Escherichia coli strain KY895 and clones were sele
cted for sensitivity to the nucleoside analogs 1-beta-D-arabinofuranosylcyt
osine (AraC, Cytarabine), 3'-azido-2',3'-dideoxythymidine (AZT, Zidovudine,
Retrovir(R)), 2',3'-dideoxyadenosine (ddA) and 2',3'-dideoxycytidine (ddC,
Zalcitabine, Hivid(R)). Thirteen mutants with increased sensitivity compar
ed to the wild-type Dm-dNK were isolated from a relatively small pool of le
ss than 10,000 clones. Eight mutant Dm-dNKs increased the sensitivity of KY
895 to more than one analog, and two of these mutants even to all four nucl
eoside analogs. Surprisingly, the mutations did not map to the five regions
which are highly conserved among deoxyribonucleoside kinases. The molecula
r background of improved sensitivity was characterized for the double-mutan
t MuD (N45D, N64D), where the LD100 value of transformed KY895 decreased 31
6-fold for AZT and more than Ii-fold for ddC when compared to wild-type Dm-
dNK. Purified recombinant MuD displayed higher K-m values for the native su
bstrates than wild-type Dm-dNK and the V-max values were substantially lowe
r. On the other hand, the K-m and V-max values for AZT and the K-m value fo
r ddC were nearly unchanged between MuD and wild-type Dm-dNK. Additionally,
a decrease in feedback inhibition of MuD by thymidine triphosphate (TTP) w
as found. This study demonstrates how high-frequency mutagenesis combined w
ith a parallel selection for desired properties provides an insight into th
e structure-function relationships of the multisubstrate kinase from D. mel
anogaster. At the same time these mutant enzymes exhibit properties useful
in biotechnological and medical applications. (C) 2000 Academic Press.