Cytosolic thymidine kinase 1, TK1, is a well known cell-cycleregulated enzyme of importance in nucleotide metabolism as well as an activator of antiviral and anticancer drugs such as 3-azido-3-deoxythymidine (AZT). We have now determined the structures of the TK1 family, the human and Ureaplasma urealyticum enzymes, in complex with the feedback inhibitor dTTP. The TK1s have a tetrameric structure in which each subunit contains an ␣͞␤-domain that is similar to ATPase domains of members of the RecA structural family and a domain containing a structural zinc. The zinc ion connects ␤-structures at the root of a ␤-ribbon that forms a stem that widens to a lasso-type loop. The thymidine of dTTP is hydrogen-bonded to main-chain atoms predominantly coming from the lasso loop. This binding is in contrast to other deoxyribonucleoside kinases where specific interactions occur with side chains. The TK1 structure differs fundamentally from the structures of the other deoxyribonucleoside kinases, indicating a different evolutionary origin.crystal structures ͉ deoxynucleotide metabolism ͉ prodrug activation

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.

SummaryUreaplasma urealyticum ( U. urealyticum ), belonging to the class Mollicutes, is a human pathogen colonizing the urogenital tract and causes among other things respiratory diseases in premature infants. We have studied the salvage of pyrimidine deoxynucleosides in U. urealyticum and cloned a key salvage enzyme, thymidine kinase (TK) from U. urealyticum . Recombinant Uu -TK was expressed in E. coli , purified and characterized with regards to substrate specificity and feedback inhibition. Uu -TK efficiently phosphorylated thymidine (dThd) and deoxyuridine (dUrd) as well as a number of pyrimidine nucleoside analogues. All natural ribonucleoside/deoxyribonucleoside triphosphates, except dTTP, served as phosphate donors, while dTTP was a feedback inhibitor. The level of Uu -TK activity in U. urealyticum extracts increased upon addition of dUrd to the growth medium. Fluoropyrimidine nucleosides inhibited U. urealyticum and M. pneumoniae growth and this inhibitory effect could be reversed by addition of dThd, dUrd or deoxytetrahydrouridine to the growth medium. Thus, the mechanism of inhibition was most likely the depletion of dTTP, either via a blocked thymidine kinase reaction and/or thymidylate synthesis step and these metabolic reactions should be suitable targets for antimycoplasma chemotherapy.