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Thymidylate kinase (TMPK, EC2.7.4.9) is the enzyme that converts deoxythymidine monophosphate (dTMP) to deoxythymidine diphosphate (dTDP) in the synthesis of dTTP, an essential building block of DNA. To date, there is only one gene (

Thymidylate synthase (TS), found in all organisms, is an essential enzyme responsible for the de novo synthesis of deoxythymidine monophosphate. The TS active sites of the protozoal parasite Cryptosporidium hominis and human are relatively conserved. Evaluation of antifolate compound 1 and its R-enantiomer 2 against both enzymes reveals divergent inhibitor selectivity and enzyme stereospecificity. To establish how C. hominis and human TS (ChTS and hTS) selectively discriminate 1 and 2, respectively, we determined crystal structures of ChTS complexed with 2 and hTS complexed with 1 or 2. Coupled with the previously determined structure of ChTS complexed with 1, we discuss a possible mechanism for enzyme stereospecificity and inhibitor selectivity.

Autosomal recessive mutations in the thymidine kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxythymidine monophosphates (dCMP+dTMP) to the Tk2 H126N (Tk2−/−) knock‐in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13‐day‐old Tk2−/− mice treated with dCMP+dTMP 200 mg/kg/day each (Tk2−/−200dCMP/dTMP) demonstrated that in mutant animals, the compounds raise dTTP concentrations, increase levels of mtDNA, ameliorate defects of mitochondrial respiratory chain enzymes, and significantly prolong their lifespan (34 days with treatment versus 13 days untreated). A second trial of dCMP+dTMP each at 400 mg/kg/day showed even greater phenotypic and biochemical improvements. In conclusion, dCMP/dTMP supplementation is the first effective pharmacologic treatment for Tk2 deficiency., This work was supported by research grants from the Muscular Dystrophy Association (MH) and the Associazione Malattie Metaboliche Congenite ereditarie (AMMeC) (CG) as well as by the Arturo Estopinan TK2 Research Fund (MH and CG) and the Marriott Mitochondrial Disease Clinic Research Fund (MMDCRF) (MH). MH acknowledges support from NIH grants (P01 HD32062, R01 HD057543, and R01 HD056103 from NICHD) and the Office of Dietary Supplements, as well as U54 NS078059 from NINDS and NICHD. LCL acknowledges support from CEIBioTic‐University of Granada, RYC‐2011‐07643, and RETICEF (Spain).

Thymidylate synthetase (TS) is known to catalyse the methylation of deoxyuridine monophosphate (dUMP) for the de novo synthesis of deoxythymidine monophosphate (dTMP). Thymidine kinase (TK) catalyzes the formation of dTMP by the phosphorylation of thymidine via the pyrimidine salvage pathway. High TS and TK activities have been observed in rapidly proliferating tissues. Both TS and TK activities in human thyroid adenocarcinoma increased to approximately 2-fold that in normal thyroid tissue. The thyroid TK isozymes can be separated into two types by DEAE-cellulose column chromatography. One of them is associated with the cytosol cell fraction of rapidly proliferating cells, such as fetal or neoplastic cells. Its activity is not affected by low concentration of deoxycytidine triphosphate (dCTP), and its molecular weight and Km value for thymidine are 120 kD and 0.5 x 10(-6) M, respectively. The average activity of this cytosol-associated isozyme in thyroid adenocarcinoma was increased slightly to 2.3-fold that of normal thyroid tissue. These results obtained from the activities of TK and its isozyme may be indicative of the slow-growing property of thyroid adenocarcinoma compared to mammary or colonic adenocarcinoma, which has high activities of TK and its isozyme.

Thymidylate synthase provides an essential function for the life of cells [8]. It provides the sole de novo pathway for synthesis of deoxythymidine monophosphate dTMP, a DNA base. Thus all organisms have thymidylate synthases with the exception with 1 or 2 algal species that obtain thymidine solely by salvage pathways. Thus thymidylate synthase is a target for antiproliferative drug design [25] aimed at pathogenic organisms in which selectivity versus host strains is important and as an antiproliferative in cancer [1,15,19,20,23,26].

In mature DNA ofBacillus subtilis phage SP10c, deoxythymidine monophosphate is partially replaced by a hypermodified nucleotide. During the interval of phage replication, infected cells contained greatly reduced levels of deoxythymidine triphosphate. However, an atypical mononucleotide, tentatively identified as 5-hydroxymethyldeoxyuridine triphosphate, was present during the interval of SP10c DNA synthesis. It is proposed that the atypical mononucleotide, and not deoxythymidine triphosphate, is a substrate for SP10c DNA replication.

The synthesis of 5-ethyl-2′-deoxyuridine-5-monophosphate (EtdUrdMP) and an improved method for the purification of deoxythymidylate synthetase (TdRMP synthetase) from E. coli are described. TdRMP synthetase was inhibited competitively by EtdUrdMP (K1 = 2·2 × 10−5 M). Under similar conditions the Ki for deoxythymidine monophosphate (TdRMP) was 2·0 × 10−5 M. The possible role of EtdUrdMP in the virostatic activity of the nucleoside 5-ethyl-2′-deoxyuridine (EtdUrd) is considered.

Autosomal recessive mutations in the thymidine kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxythymidine monophosphates (dCMP+dTMP) to the Tk2 H126N (Tk2(-/-)) knock-in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13-day-old Tk2(-/-) mice treated with dCMP+dTMP 200 mg/kg/day each (Tk2(-/-200dCMP/) (dTMP)) demonstrated that in mutant animals, the compounds raise dTTP concentrations, increase levels of mtDNA, ameliorate defects of mitochondrial respiratory chain enzymes, and significantly prolong their lifespan (34 days with treatment versus 13 days untreated). A second trial of dCMP+dTMP each at 400 mg/kg/day showed even greater phenotypic and biochemical improvements. In conclusion, dCMP/dTMP supplementation is the first effective pharmacologic treatment for Tk2 deficiency.