Structural basis for substrate promiscuity of dCK.
Deoxycytidine kinase (dCK) is an essential nucleoside kinase critical for the production of nucleotide precursors for DNA synthesis. This enzyme catalyzes the initial conversion of the nucleosides deoxyadenosine (dA), deoxyguanosine (dG), and deoxycytidine (dC) into their monophosphate forms, with subsequent phosphorylation to the triphosphate forms performed by additional enzymes. Several nucleoside analog prodrugs are dependent on dCK for their pharmacological activation, and even nucleosides of the non-physiological L-chirality are phosphorylated by dCK. In addition to accepting dC and purine nucleosides (and their analogs) as phosphoryl acceptors, dCK can utilize either ATP or UTP as phosphoryl donors. To unravel the structural basis for substrate promiscuity of dCK at both the nucleoside acceptor and nucleotide donor sites, we solved the crystal structures of the enzyme as ternary complexes with the two enantiomeric forms of dA (D-dA, or L-dA), with either UDP or ADP bound to the donor site. The complexes with UDP revealed an open state of dCK in which the nucleoside, either D-dA or L-dA, is surprisingly bound in a manner not consistent with catalysis. In contrast, the complexes with ADP, with either D-dA or L-dA, adopted a closed and catalytically competent conformation. The differential states adopted by dCK in response to the nature of the nucleotide were also detected by tryptophan fluorescence experiments. Thus, we are in the unique position to observe differential effects at the acceptor site due to the nature of the nucleotide at the donor site, allowing us to rationalize the different kinetic properties observed with UTP to those with ATP.
Sabini
Elisabetta
E
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 South Ashland Avenue, Chicago, IL 60607, USA.
Hazra
Saugata
S
Ort
Stephan
S
Konrad
Manfred
M
Lavie
Arnon
A
D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D003839:Deoxyadenosines / Q000378:metabolism; D003841:Deoxycytidine / Q000378:metabolism; D003842:Deoxycytidine Kinase / Q000737:chemistry* / Q000378:metabolism*; D003849:Deoxyguanosine / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D003839:Deoxyadenosines; D003841:Deoxycytidine; D003842:Deoxycytidine Kinase; D003849:Deoxyguanosine