Structure of the substrate complex of thymidine kinase from Ureaplasma urealyticum and investigations of possible drug targets for the enzyme.
Thymidine kinases have been found in most organisms, from viruses and bacteria to mammals. Ureaplasma urealyticum (parvum), which belongs to the class of cell-wall-lacking Mollicutes, has no de novo synthesis of DNA precursors and therefore has to rely on the salvage pathway. Thus, thymidine kinase (Uu-TK) is the key enzyme in dTTP synthesis. Recently the 3D structure of Uu-TK was determined in a feedback inhibitor complex, demonstrating that a lasso-like loop binds the thymidine moiety of the feedback inhibitor by hydrogen bonding to main-chain atoms. Here the structure with the substrate deoxythymidine is presented. The substrate binds similarly to the deoxythymidine part of the feedback inhibitor, and the lasso-like loop binds the base and deoxyribose moieties as in the complex determined previously. The catalytic base, Glu97, has a different position in the substrate complex from that in the complex with the feedback inhibitor, having moved in closer to the 5'-OH of the substrate to form a hydrogen bond. The phosphorylation of and inhibition by several nucleoside analogues were investigated and are discussed in the light of the substrate binding pocket, in comparison with human TK1. Kinetic differences between Uu-TK and human TK1 were observed that may be explained by structural differences. The tight interaction with the substrate allows minor substitutions at the 3 and 5 positions of the base, only fluorine substitutions at the 2'-Ara position, but larger substitutions at the 3' position of the deoxyribose.
Kosinska
Urszula
U
Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Sweden.
Carnrot
Cecilia
C
Eriksson
Staffan
S
Wang
Liya
L
Eklund
Hans
H
D001665:Binding Sites; D016503:Drug Delivery Systems; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D013937:Thymidine Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D013942:Thymine Nucleotides / Q000096:biosynthesis; D016990:Ureaplasma urealyticum / Q000201:enzymology*
D013942:Thymine Nucleotides; D013937:Thymidine Kinase; C024157:thymidine 5'-triphosphate