Thymidylate kinase is a ubiquitous enzyme of about 25 kd and is important in the dttp synthesis pathway for dna synthesis. Thymidylate kinase an overview sciencedirect topics. Herpes simplex virus type 1 thymidine kinase exhibits a strikingly broad substrate specificity. Catalytic mechanism and structure of viral flavindependent. Pa530010 targets thymidylate kinase in wb applications and shows reactivity with human samples. Thymidylate medical definition merriamwebster medical. Human thymidine kinase can functionally replace herpes. In the aim of finding a new target, the thymidylate tmp kinase from vaccinia virus, the prototype of orthopoxvirus, has been overexpressed in escherichia coli after cloning the gene a48r. Tmk thymidylate kinase ectromelia virus strain moscow. Previously, deletion of either the tk gene or vgf genes was shown to. Lid region because of the deletion of residues in vacctmpk fig. Us20110014165a1 thymidylate kinase mutants and uses thereof. Random mutagenesis of the thymidine kinase gene of varicellazoster virus.
Fingeroth 1, 2, 3, divisions of infectious disease, 1 experimental medicine, 2 beth israel deaconess medical center, harvard medical school, 3 department of pathology. Random mutagenesis of the thymidine kinase gene of. Thymidylate kinase recombinant protein, prosci vwr. Dtymk rabbit antihuman, polyclonal, invitrogen 100 l. Unlike tk2, which is not modulated by the cell cycle, tk1 expression and activity is regulated in a cell cycledependent manner, accumulating during g1phase to peak levels in sphase before being degraded prior to cell.
Stabilization of active site dynamics leads to increased. However, a gradual reduction in plaque size and number was. Dominant negative selection of vaccinia virus using a thymidine. Database searching for thymidine and thymidylate kinase. All analyses were performed using jmp software sas institute inc. Thymidylate kinase tmpk catalyzes the conversion of thymidine monophosphate to thymidine diphosphate, which is an essential step for dttp synthesis. A mutant herpes simplex virus type 1 thymidine kinase protein, wherein the glutamine at position 125 of the wild type of said kinase is changed by sitespecific mutation to another amino acid, wherein said mutant protein has improved nucleoside analog metabolizing activity hut has reduced thymidine phosphorylation activity relative to the wildtype protein. Thymidylate synthase ts is an e2f1regulated enzyme that is essential for dna synthesis and repair. Pdf a novel viral thymidylate kinase with dual kinase.
Human thymidylate kinase htmpk conversion of the antihiv prodrug 3. Oncolytic efficacy of thymidine kinasedeleted vaccinia virus strain. Arg93, which is part of the drx motif of thymidylate kinases, is the only. Epsteinbarr virus thymidine kinase is a centrosomal resident precisely localized to the periphery of centrioles michael b.
The structures of tk complexed with adp at the atp. Although the data suggest strongly that some of these proteins are indeed virusdetermined, e. Aug 28, 2015 nucleotide phosphorylation is a key step in dna replication and viral infections, since suitable levels of nucleotide triphosphates pool are required for this process. A cysteine residue is involved in the catalytic mechanism it covalently binds the 5,6dihydrodump intermediate. Pdf virus encodes an active thymidylate kinase that nts a. Data were fitted using microcal origin software to yield. Design of inhibitors of thymidylate kinase from variola virus as new selective drugs against smallpox. O2methoxyethyl nucleosides are not phosphorylated or. The structures of thymidine kinase from herpes simplex. The vaccinia virus genes encoding ribonucleotide reductase, thymidine kinase, thymidylate kinase and dutpase are transcribed early during the infection cycle to provide a large amount of dna precursors. Thymidylate kinase dtymk is a ubiquitous enzyme of about 25 kd which belongs to thymidylate kinase family. The large substrate specificity is explained by the presence of a. Epsteinbarr virus thymidine kinase is a centrosomal resident. Purified, bacterially expressed vzv tk was characterized with regard to nterminal amino acid sequence, pi value, ph optimum, metal ion requirement, phosphate donor and acceptor specificity, and inhibition by dttp.
Structural and functional analyses suggest that the cdna codes for authentic human dtmp kinase. Human herpesvirus 8 gene encodes a functional thymidylate. We overexpressed the tmk orf454 domain tmkwssv and its specific activity was measured with dtmp and dtdp as phosphate acceptors. An enzymatic assay for herpes virus simplex type 1 thymidine kinase hsvtk that was sensitive enough to quantitate intracellular levels of enzyme transiently expressed after transfection of hsvtk vectors. C l talarico, w c phelps, and k k biron division of virology, wellcome.
A copy of the orf, generated by polymerase chain reaction. Tmk thymidylate kinase rabbitpox virus strain utrecht. The gene has been shown to encode an active dtmp kinase 23 kda and to complement a temperature. Substrate specificity of vaccinia virus thymidylate kinase. Hhv8 replication can be selectively inhibited by several antiherpesvirus. Ts protein and mrna levels are elevated in many human cancers, and high ts levels have been correlated with poor prognosis in patients with colorectal, breast, cervical, bladder, kidney, and nonsmall cell lung cancers. A novel viral thymidylate kinase with dual kinase activity. The vaccinia virus thymidylate kinase gene has been cloned and identified by. A thymidine kinasedeleted bovine herpesvirus 5 establishes latent. Considering this threat and the nonexistence of effective chemotherapy, we propose the enzyme thymidylate kinase from variola virus vartmpk as a potential target to the drug design against. Pdf structure of vaccinia virus thymidine kinase in.
Crystal structure of vaccinia virus thymidylate kinase bound to tdp. Mutated herpes simplex virus type 1 thymidine kinases and. After infection with virus, cancer cells expressed markers for. Herpes simplex virus thymidine kinase enzymatic assay in. The pa530010 immunogen is recombit fragment corresponding to a region within amino acids 1 and 207 of human thymidylate kinase.
Clicking the images or links will redirect you to a website hosted by benchsci that provides thirdparty scientific content. Neither the content nor the benchsci technology and processes for selection have been evaluated by us. These functions include those of thymidine kinase, deoxycytidine kinase, thymidylate kinase, deoxycytidylate deaminase, ribonucleotide reductase and dna polymerase. Molecular characterization of thymidine kinase and thymidylate kinase in ureaplasma urealyticum and bacillus anthracis. High levels of thymidylate synthase may be involved in how certain types of cancer form and respond to treatment. Two different classes of thymidine kinases have been identified and are included in this super family. We have solved the crystal structures of vaccinia virus thymidylate kinase bound to tdp or brivudin monophosphate.
Analysis of the alignment of the tks of the first family highlighted three strongly conserved segments. The engineering involves replacing the native thymidine kinase vvtk gene, in a wyeth strain vaccinia backbone, with the herpes simplex virus thymidine kinase hsvtk gene, which allows for. The ability of thymidine kinase tkdeleted recombinant bovine herpesvirus 5. Purified, bacterially expressed vzv tk was characterized with regard to n. Ts also functions as a rna binding protein and its protein levels are linked to chemoresistance in cancer. International audiencestructurebased drug design methods were used to search for novel inhibitors of herpes simplex virus type 1 hsv1 thymidine kinase and mycobacterium tuberculosis thymidylate kinase. Analysis of the thymidine kinase genes from acyclovirresistant mutants of varicellazoster virus isolated from patients with aids. On the other hand, removal of the methyl group dump decreased the affinity. Although the viral and human enzymes have similar sequences 42% identity, they.
Cellfree synthesis of enzymatically active vaccinia virus thymidine kinase. Vaccinia virus encodes a thymidine kinase gene tk that, when deleted. The other 11 mutations resulted in reduction of both tk and thymidylate kinase activities or only thymidylate kinase activity. Phylogenetic analysis of viral and cellular thymidine and thymidylate kinases was performed using computerassisted methods. The invention relates to a composition comprising a stably integrating delivery vector. Certain viruses also have genetic information for expression of viral thymidine. Characterization of pyrimidine deoxyribonucleoside kinase thymidine kinase and thymidylate kinase as a multifunctional enzyme in cells transformed by herpes simplex virus type 1 and in cells infected with mutant strains of herpes simplex virus. Substrate diversity of herpes simplex virus thymidine kinase. Epsteinbarr virus thymidine kinase is a centrosomal. In marked contrast with wildtype hsv1 tk, which displays both thymidine kinase and thymidylate kinase activities, the hsv1 tkq125n mutant was unable to phosphorylate pyrimidine nucleoside. The function of dtmp kinase in eukaryotes comes from the study of a cell cycle mutant, cdc8, in saccharomyces cerevisiae.
Structurebased drug design methods were used to search for novel inhibitors of herpes simplex virus type 1 hsv1 thymidine kinase and mycobacterium tuberculosis thymidylate kinase. Thymidylate synthase is the enzyme responsible for the conversion by methylation of deoxyuridine monophosphate, dump, to thymidine monophosphate which is a deoxyribose called either tmp or dtmp which after conversion into thymidine triphosphate, ttp, is incorporated in dna. Being the exclusive source of dtmp, investigators have. Substrate specificity of vaccinia virus thymidylate kinase topalis. Herpesvirus infection increases at least six enzymatic functions related to dna synthesis, and these may relate to the formation of new proteins determined by viral genes. Thymidylate synthetase how is thymidylate synthetase. A chimeric poxvirus with j2r thymidine kinase deletion shows. Usually, tk gene deletion leads to deficient virus replication in neurons and reduced. Although several kinases, like sr protein kinases srpks 1 and 2, clksty, dualspecificity tyrosineregulated kinase, crkrs, dna topoisomerase i, glycogen synthase kinase 3 and akt, have already been proven to phosphorylate sr proteins 1924, the signaltransduction pathways that regulate option splicing remain improperly comprehended. The large substrate specificity is explained by the presence of a canal connecting the edge of the dimer interface to the tmp base binding pocket, canal not found in the human homolog by similarity. Tmk thymidylate kinase camelpox virus strain cms tmk. Quantification and analysis of thymidine kinase expression from. Thymidylate synthase is an enzyme of about 30 to 35 kda in most species except in protozoan and plants where it exists as a bifunctional enzyme that includes a dihydrofolate reductase domain.
Thymidylate kinase is critical for dna repair via atm. Analysis of the thymidine kinase genes from acyclovir. Tmk thymidylate kinase vaccinia virus strain tian tan. Pdf modifying human thymidylate kinase to potentiate. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic 4pl curvefit. It is capable of phosphorylating deoxythymidine and deoxyuridine as does human thymidine kinase, deoxycytidine as does human deoxycytidine kinase, the cytosolic kinase whose amino acid sequence it most closely resembles, and thymidylate as does human thymidylate kinase. Herpes simplex virus type 1 hsv 1 thymidine kinase tk exhibits an extensive substrate diversity for nucleobases and sugar moieties, in contrast to other tks. It is present in two forms in mammalian cells, tk1 and tk2. Thymidine kinase is an enzyme, a phosphotransferase a kinase. The vaccinia virus thymidylate kinase gene has been cloned and identified by homology with yeast thymidylate kinase 42% identity.
Deletion of the thymidine kinase gene induces complete attenuation. African swine fever virus asfv is the etiological agent of a contagious and often lethal viral disease of. The nucleic acid substrate for ts is uridine monophosphate dump. Human herpesvirus 1 hhv1 human herpes simplex virus 1. The results were consistent with the supposition that tk deletion would. Thymidine kinase from herpes simplex virus type 1 tk was crystallized in an n. This process is catalyzed by thymidylate synthase ts or tyms, a homodimer composed of two 30 kda subunits. There are two known thymidine kinases, cytoplasmic thymidine kinase 1 tk1 and mitochondrial thymidine kinase 2 tk2 1,2.
For epsteinbarr virus ebv tk, removal of the nterminal domain does not. The predicted polypeptide shares 42% amino acid identity over a 200 amino acid region with saccharomyces cerevisiae thymidylate kinase tmpk and has low homology with herpes simplex virus deoxypyrimidine kinase. Structure of vaccinia virus thymidine kinase in complex. In contrast to the conventional vaccinia thymidine kinase tk selection that. As a member of the wwpdb, the rcsb pdb curates and annotates pdb data according to agreed upon standards. White spot syndrome virus wssv genome contains an open reading frame orf454 that encodes a thymidine kinase and tmk domains in a single polypeptide. A vaccinia virus open reading frame orf previously predicted to encode thymidylate kinase tmpk is shown to encode an active enzyme. Thus, tk deletion had no apparent effect on the frequency of latent infection but. Thymidylate kinases are essential enzymes with roles in dna synthesis and repair and have been the target of drug development for antimalarials, antifungals, hiv treatment, and cancer therapeutics. Thymidyne monophosphate kinase tmk is the enzyme in the junction of. Virology 31, 120128 1967 a virus specific thymidine kinase in bhk21 cells infected with herpes simplex virus h.
Thymidine kinase 1 tk1, human adenylate kinase 1 ak1, human guanylate kinase 1 gk1, human deoxythymidylate kinase thymidylate kinase dtymk were obtained from prospec israel. It catalyzes the addition of a methyl group, ch3, to the dump to produce thymidine monophosphate dtmp. Design of inhibitors of thymidylate kinase from variola. We focus here on vaccinia virus thymidylate kinase vvtmp kinase.
Alphaherpesvirus thymidine kinase genes mediate viral. Some herpes viruses encode only 1 enzyme catalyzing both. Certain viruses also have genetic information for expression of viral thymidine kinases. This substrate diversity is the crucial molecular basis of selective antiviral and suicide gene therapy. Tk thymidine kinase human herpesvirus 1 hhv1 tk gene. Conservative mutations of glutamine125 in herpes simplex virus type 1 thymidine kinase result in a ganciclovir kinase with minimal deoxypyrimidine kinase activities. Systemic cancer therapy with a tumorselective vaccinia virus. Recently we constructed a homology model of the enzyme thymidylate kinase from variola virus vartmpk and proposed it as a new target to the drug design against smallpox. The rcsb pdb also provides a variety of tools and resources. A virusspecific thymidine kinase in bhk21 cells infected. Although the data suggest strongly that some of these proteins are indeed virus determined, e. Human herpesvirus 8 hhv8 is a gammaherpesvirus that is related to herpesvirus saimiri hvs and rhesus rhadinovirus rrv. Vaccinia virus is a useful tool in biomedical research and recombinant viruses are.
The equine herpes virus 4 thymidine kinase is a better. The varicellazoster virus vzv thymidine kinase tk ec 2. Thymidylate synthase how is thymidylate synthase abbreviated. May 04, 2020 since thymidylate synthase is an enzyme, it catalyzes reactions to happen more quickly than they would in its absence. Human deoxycytidine kinase dck, human uridine monophosphate cytidine monophosphate kinase umpcmpk kinases were obtained from novocib france. Cpf, replication factor c rfc3, thymidylate synthetase tyms, a putative mitotic checkpoint. Thymihdihlayt sinthays a protein involved in making and repairing dna molecules inside cells that carry genetic information and pass it on from parent to child. Thymidylate kinase dtymk is a ubiquitous enzyme of about 25 kd which belongs to thymidylate kinase family this recombinant protein can be used for biological assays. Thymidine kinase from herpesvirus is a subfamily of thymidine kinases its presence in herpesvirusinfected cells is used to activate a range of antivirals against herpes infection, and thus specifically. Many acyclovirresistant herpes simplex virus mutants from clinical isolates have. Poxvirus tmp kinase is able to phosphorylate dtmp, dump and also dgmp from any purine and pyrimidine nucleoside triphosphate. The thymidine kinase tk encoded by epsteinbarr virus ebv differs not only. Alphaherpesvirus thymidine kinase tk genes are virulencerelated genes. Pilger bd, perozzo r, alber f, wurth c, folkers g, scapozza l.