A mathematical model of human thymidine kinase 2 activity

The mitochondrial enzyme thymidine kinase 2 (TK2) phosphorylates deoxythymidine (dT) and deoxycytidine (dC) to form dTMP and dCMP, which in cells rapidly become the negative-feedback end-products dTTP and dCTP. TK2 kinetic activity exhibits Hill coefficients of ～0.5 (apparent negative cooperativity) for dT and ～1 for dC. We present a mathematical model of TK2 activity that is applicable if TK2 exists as two monomer forms in equilibrium.     

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.     

The crystal structure of Mycobacterium tuberculosis thymidylate kinase in complex with 3'-azidodeoxythymidine monophosphate suggests a mechanism for competitive inhibition

Tuberculosis (TB) is the primary cause of mortality among infectious diseases. Mycobacterium tuberculosis thymidylate kinase (TMPK(Mtub)) catalyzes the ATP-dependent phosphorylation of deoxythymidine 5'-monophosphate (dTMP). Essential to DNA replication, this enzyme represents a promising target for developing new drugs against TB, because the configuration of its active site is unique within the TMPK family. Indeed, it has been proposed that, as opposed to other TMPKs, catalysis by TMPK(Mtub) necessitates the transient binding of a magnesium ion coordinating the phosphate acceptor. Moreover, 3'-azidodeoxythymidine monophosphate (AZTMP) is a competitive inhibitor of TMPK(Mtub), whereas it is a substrate for human and other TMPKs. Here, the crystal structures of TMPK(Mtub) in complex with deoxythymidine (dT) and AZTMP were determined to 2.1 and 2.0 A resolution, respectively, and suggest a mechanism for inhibition. The azido group of AZTMP perturbs the induced-fit mechanism normally adopted by the enzyme. Magnesium is prevented from binding, and the resulting electrostatic environment precludes phosphoryl transfer from occurring. Our data provide a model for drug development against tuberculosis.     

Structural features of two distinct molecular complexes of copper(II) cationic porphyrin and deoxyribonucleotides

The associations of the water-soluble cationic copper(II)-5,10,15,20-meso-tetrakis(4-N-methylpyridyl) porphyrin (CuP) with d(pT)9 oligothymidylate and its building blocks deoxythymidine (dT) and deoxythymidine 5'-monophosphate (dTMP) were investigated by spectrophotometric titration [absorption, nanosecond transient resonance Raman (ns-RR) and picosecond time-resolved resonance Raman (ps-TR3) spectroscopies] to elucidate the structural requirements for the CuP exciplex formation in molecular complexes with unchained mononucleotides. In the d(pT)9 a factor analysis and global fit of the CuP absorption spectra revealed the formation of a single spectral species attributable to a 1 : 1 CuP. d(pT)9 complex throughout a wide range of d(pT)9/CuP ratios (0-10). Using ps-TR3 spectroscopy, the CuP. d(pT)9 complex was shown to be fully responsible for exciplex formation. In contrast, CuP mixed with dTMP ([dTMP]/[CuP] < 3000) yielded two spectroscopically distinct types of molecular complexes with 1 : 1 (CuP. dTMP) and 1 : 2 (CuP. (dTMP)2) (or even higher for [dTMP]/[CuP] > 3000) stoichiometry, the latter being spectroscopically identical to the CuP. d(pT)9 and providing a microenvironment favorable for exciplex formation to the same extent as the oligothymidylate. On the other hand, the 1 : 1 CuP. dTMP complex (prevailing for [dTMP]/[CuP] < 100) yielded no exciplex features. Similar behavior was observed for the CuP complexed with dT. To explain the difference in the ability of the CuP. dTMP and CuP. (dTMP)2 species to form the exciplex, two types of molecular complexes were suggested and discussed, differing in the orientation of the thymine planes with respect to the porphyrin macrocycle.     

Toward a structural understanding of the dehydratase mechanism.

dTDP-D-glucose 4,6-dehydratase (RmlB) was first identified in the L-rhamnose biosynthetic pathway, where it catalyzes the conversion of dTDP-D-glucose into dTDP-4-keto-6-deoxy-D-glucose. The structures of RmlB from Salmonella enterica serovar Typhimurium in complex with substrate deoxythymidine 5'-diphospho-D-glucose (dTDP-D-glucose) and deoxythymidine 5'-diphosphate (dTDP), and RmlB from Streptococcus suis serotype 2 in complex with dTDP-D-glucose, dTDP, and deoxythymidine 5'-diphospho-D-pyrano-xylose (dTDP-xylose) have all been solved at resolutions between 1.8 A and 2.4 A. The structures show that the active sites are highly conserved. Importantly, the structures show that the active site tyrosine functions directly as the active site base, and an aspartic and glutamic acid pairing accomplishes the dehydration step of the enzyme mechanism. We conclude that the substrate is required to move within the active site to complete the catalytic cycle and that this movement is driven by the elimination of water. The results provide insight into members of the SDR superfamily.     

Cell cycle regulation and novel structural features of thymidine kinase,an essential enzyme in Trypanosoma brucei

Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ‐phosphate of ATP to 2′‐deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C‐terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine‐derived nucleotides. In addition, we report the X‐ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design.     

The First Crystal Structure of a dTTP-bound Deoxycytidylate Deaminase Validates and Details the Allosteric-Inhibitor Binding Site

Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. Here we present the first crystal structure of a dTTP-bound deoxycytidylate deaminase from the bacteriophage S-TIM5, confirming that this inhibitor binds to the same site as the dCTP activator. The molecular details of this structure, complemented by structures apo- and dCMP-bound, provide insights into the allosteric mechanism. Although the positioning of the nucleoside moiety of dTTP is almost identical to that previously described for dCTP, protonation of N3 in deoxythymidine and not deoxycytidine would facilitate hydrogen bonding of dTTP but not dCTP and may result in a higher affinity of dTTP to the allosteric site conferring its inhibitory activity. Further the functional group on C4 (O in dTTP and NH₂ in dCTP) makes interactions with nonconserved protein residues preceding the allosteric motif, and the relative strength of binding to these residues appears to correspond to the potency of dTTP inhibition. The active sites of these structures are also uniquely occupied by dTMP and dCMP resolving aspects of substrate specificity. The methyl group of dTMP apparently clashes with a highly conserved tyrosine residue, preventing the formation of a correct base stacking shown to be imperative for deamination activity. The relevance of these findings to the wider zinc-dependent cytidine deaminase family is also discussed.     

Deoxythymidine 3', 5'-di-p-nitrophenyl phosphate as a synthetic substrate for bovine pancreatic deoxyribonuclease.

Bovine pancreatic deoxyribonuclease liberates p-nitrophenol from the 3'-group of deoxythymidine 3', 5'-di-p-nitrophenyl phosphate. A similar hydrolysis occurs with deoxythymidine 3'-p-nitrophenyl phosphate 5'-phsophate, but the rate is less than 2% of that with the di-p-nitrophenyl ester. The rate of formation of the p-nitrophenol, measured spectrophotometrically at 400 nm, varies linearly with DNase concentration. The binding of the substrate is not strong (K-m(app) in the 10 mM range), but the hydrolysis is rapid; 1 mug of DNase (free from other phosphodiesterases) can be assayed in 3 min after addition to a 10 mM substrate solution at pH 7.2, 10mM in MnCl2, and 1mM in CaCl2. All four bovine pancreatic DNases (A,B,C, and D) show the same relative activities toward DNA and toward the di-p-nitrophenyl ester; both activities are lost when DNase is inactivated by iodoacetate or by trypsin. The specificity of DNase toward the di-p-nitrophenyl substrate is different from that which has been established for the enzyme's predominant action on DNA or synthetic oligonucleotides, where a monoesterified phosphate group is formed at the 5'-position.     

Cyanamide mediated syntheses under plausible primitive earth conditions

Summary When an aqueous solution (pH 7.0) of deoxythymidine 5-phosphate, 4-amino-5-imidazolecarboxamide and cyanamide was dried and heated for 18 h at 60°C, P¹, P²-dideoxythymidine 5-pyrophosphate (I) was formed in a 58% yield. Oligonucleotides were not detected in the reaction product. Under conditions employed in the above reaction, (I) was shown to be stable. In prebiotic polymerization reactions employing deoxythymidine 5-triphosphate as the polymerizing species, (I) could therefore function as a primer and minimize the formation of cyclic nucleotides.Abbreviations dT deoxythymidine - dTMP deoxythymidine 5-phosphate - dTppT P¹, P²-dideoxythymidine 5-pyrophosphate - dTTP deoxythymidine 5-triphosphate - AICA 4-amino-5-imidazolecarboxamide     

Enzymes of deoxythymidine triphosphate biosynthesis in Neurospora crassa mitochondria.

Intact mitochondria of Neurospora crassa incorporate deoxythymidine 5'-monophosphate (dTMP) into deoxyribonucleic acid but not the label from (methyl-3H) deoxythymidine. Mitochondrial homogenates contain deoxythymidylate kinase (EC 2.7.4.9), deoxycytidylate aminohydrolase (dCMP deaminase) (EC 3.5.4.12), and thymidylate synthetase (EC 2.1.1b), but not thymidine kinase (EC 2.7.1.21) activity. dTMP kinase is loosely bound to the mitochondrial membrane and is solubilized by 0.4 M KCl in mitochondrial homogenates, the dCMP aminohydrolase deaminase) is bound to the inner membrane and is not solubilized by 0.4 M KCl. dTMP synthetase activity is found in the 2,000 times g particulate fractions by homogenization of mitochondria in 0.4 M KCl. The dCMP deaminase activity found in the particulate fraction of the inner membrane is efficiently regulated by the products of the pathway: deoxycytidine 5'-triphosphate activates whereas deoxythymidine 5'-triphosphate inhibits, as found for the soluble enzyme from other sources. These data indicate that mitochondria of N. crassa contain specific enzymes for the biosynthesis of deoxythymidine triphosphate.     

Structural and mechanistic studies on carboxymethylproline synthase (CarB), a unique member of the crotonase superfamily catalyzing the first step in carbapenem biosynthesis

The first step in the biosynthesis of the medicinally important carbapenem family of beta-lactam antibiotics is catalyzed by carboxymethylproline synthase (CarB), a unique member of the crotonase superfamily. CarB catalyzes formation of (2S,5S)-carboxymethylproline [(2S,5S)-t-CMP] from malonyl-CoA and l-glutamate semialdehyde. In addition to using a cosubstrate, CarB catalyzes C-C and C-N bond formation processes as well as an acyl-coenzyme A hydrolysis reaction. We describe the crystal structure of CarB in the presence and absence of acetyl-CoA at 2.24 A and 3.15 A resolution, respectively. The structures reveal that CarB contains a conserved oxy-anion hole probably required for decarboxylation of malonyl-CoA and stabilization of the resultant enolate. Comparison of the structures reveals that conformational changes (involving His(229)) in the cavity predicted to bind l-glutamate semialdehyde occur on (co)substrate binding. Mechanisms for the formation of the carboxymethylproline ring are discussed in the light of the structures and the accompanying studies using isotopically labeled substrates; cyclization via 1,4-addition is consistent with the observed labeling results (providing that hydrogen exchange at the C-6 position of carboxymethylproline does not occur). The side chain of Glu(131) appears to be positioned to be involved in hydrolysis of the carboxymethylproline-CoA ester intermediate. Labeling experiments ruled out the possibility that hydrolysis proceeds via an anhydride in which water attacks a carbonyl derived from Glu(131), as proposed for 3-hydroxyisobutyryl-CoA hydrolase. The structural work will aid in mutagenesis studies directed at altering the selectivity of CarB to provide intermediates for the production of clinically useful carbapenems.     

The structural mechanism for half-the-sites reactivity in an enzyme, thymidylate synthase, involves a relay of changes between subunits.

Thymidylate synthase (TS), a half-the-sites reactive enzyme, catalyzes the final step in the de novo biosynthesis of deoxythymidine monophosphate, dTMP, required for DNA replication. The cocrystal structure of TS from Pneumocystis carinii (PcTS), a new drug target for an important pathogen, with its substrate, deoxyuridine monophosphate (dUMP), and a cofactor mimic, CB3717, was determined. The structure, solved at 2.6 A resolution, shows an asymmetric dimer with two molecules of the substrate dUMP bound yet only one molecule of cofactor analogue bound. The structural evidence reveals that upon binding cofactor analogue and forming a covalent bond from the nucleophilic cysteine to the substrate, dUMP, at one active site, PcTS undergoes a conformational change that renders the opposite monomer incapable of forming a covalent bond or binding a molecule of cofactor analogue. The communication pathway between the two active sites is evident, allowing a structural definition of the basis of half-the-sites reactivity for thymidylate synthase and providing an example of such a mechanism for other half-the-sites reactive enzymes.     

Mutation of herpesvirus thymidine kinase to generate ganciclovir-specific kinases for use in cancer gene therapies

Understanding the functional and mechanistic properties of the multi-substrate herpes simplex virus type-1 thymidine kinase (HSV-1 TK) remains critical to defining its role as a major pharmacological target in herpesvirus and gene therapies for cancer. An inherent limitation of the activity of HSV-TK is the >70-fold difference in the K(m)s for phosphorylation of thymidine over the pro-drug ganciclovir (GCV). To engineer an HSV-1 TK isoform that is specific for GCV as the preferred substrate, 16 site-specific mutants were generated. The mutations were concentrated at conserved residues involved in nucleoside base binding, Gln125 and near sites 3 and 4 involved in catalysis and substrate binding. The substrate preferences of each mutant enzyme were compared with wild-type HSV-1 TK. One mutant, termed Q7530 TK, had a lower K(m) for GCV than thymidine. Expression of the Q7530 TK in tumor cells indicated comparable metabolism to and improved sensitivity to GCV over wild-type HSV-1 TK, with minimal thymidine phosphorylation activity. A molecular modeling simulation of the different HSV-1 TK active-sites was done for GCV and thymidine binding. It was concluded that mutations at Gln125 and near site 4, especially at Ala168, were responsible for loss of deoxypyrimidine substrate binding.     

Exploring the active site of herpes simplex virus type-1 thymidine kinase by X-ray crystallography of complexes with aciclovir and other ligands

Antiherpes therapies are principally targeted at viral thymidine kinases and utilize nucleoside analogs, the triphosphates of which are inhibitors of viral DNA polymerase or result in toxic effects when incorporated into DNA. The most frequently used drug, aciclovir (Zovirax), is a relatively poor substrate for thymidine kinase and high-resolution structural information on drugs and other molecules binding to the target is therefore important for the design of novel and more potent chemotherapy, both in antiherpes treatment and in gene therapy systems where thymidine kinase is expressed. Here, we report for the first time the binary complexes of HSV-1 thymidine kinase (TK) with the drug molecules aciclovir and penciclovir, determined by X-ray crystallography at 2.37 Å resolution. Moreover, from new data at 2.14 Å resolution, the refined structure of the complex of TK with its substrate deoxythymidine (R = 0.209 for 96% of all data) now reveals much detail concerning substrate and solvent interactions with the enzyme. Structures of the complexes of TK with four halogen-containing substrate analogs have also been solved, to resolutions better than 2.4 Å. The various TK inhibitors broadly fall into three groups which together probe the space of the enzyme active site in a manner that no one molecule does alone, so giving a composite picture of active site interactions that can be exploited in the design of novel compounds. Proteins 32:350–361, 1998. © 1998 Wiley-Liss, Inc.     

Kinetics and crystal structure of the wild-type and the engineered Y101F mutant of Herpes simplex virus type 1 thymidine kinase interacting with (North)-methanocarba-thymidine

Kinetic and crystallographic analyses of wild-type Herpes simplex virus type 1 thymidine kinase (TK(HSV1)) and its Y101F-mutant [TK(HSV1)(Y101F)] acting on the potent antiviral drug 2'-exo-methanocarba-thymidine (MCT) have been performed. The kinetic study reveals a 12-fold K(M) increase for thymidine processed with Y101F as compared to the wild-type TK(HSV1). Furthermore, MCT is a substrate for both wild-type and mutant TK(HSV1). Its binding affinity for TK(HSV1) and TK(HSV1)(Y101F), expressed as K(i), is 11 microM and 51 microM, respectively, whereas the K(i) for human cytosolic thymidine kinase is as high as 1.6 mM, rendering TK(HSV1) a selectivity filter for antiviral activity. Moreover, TK(HSV1)(Y101F) shows a decrease in the quotient of the catalytic efficiency (k(cat)/K(M)) of dT over MCT corresponding to an increased specificity for MCT when compared to the wild-type enzyme. Crystal structures of wild-type and mutant TK(HSV1) in complex with MCT have been determined to resolutions of 1.7 and 2.4 A, respectively. The thymine moiety of MCT binds like the base of dT while the conformationally restricted bicyclo[3.1.0]hexane, mimicking the sugar moiety, assumes a 2'-exo envelope conformation that is flatter than the one observed for the free compound. The hydrogen bond pattern around the sugar-like moiety differs from that of thymidine, revealing the importance of the rigid conformation of MCT with respect to hydrogen bonds. These findings make MCT a lead compound in the design of resistance-repellent drugs for antiviral therapy, and mutant Y101F, in combination with MCT, opens new possibilities for gene therapy.     

Mispairs with Watson‐Crick base‐pair geometry observed in ternary complexes of an RB69 DNA polymerase variant

Recent structures of DNA polymerase complexes with dGMPCPP/dT and dCTP/dA mispairs at the insertion site have shown that they adopt Watson‐Crick geometry in the presence of Mn²⁺ indicating that the tautomeric or ionization state of the base has changed. To see whether the tautomeric or ionization state of base‐pair could be affected by its microenvironment, we determined 10 structures of an RB69 DNA polymerase quadruple mutant with dG/dT or dT/dG mispairs at position n‐1 to n‐5 of the Primer/Template duplex. Different shapes of the mispairs, including Watson‐Crick geometry, have been observed, strongly suggesting that the local environment of base‐pairs plays an important role in their tautomeric or ionization states.     

The insulin receptor tyrosine kinase domain in a chimaeric epidermal growth factor-insulin receptor generates Ca2+ signals through the PLC-gamma1 pathway.

The receptors for insulin (IR) and epidermal growth factor (EGFR) are members of the tyrosine kinase receptor (TKR) family. Despite homology of their cytosolic TK domains, both receptors induce different cellular responses. Tyrosine phosphorylation of insulin receptor substrate (IRS) molecules is a specific IR post-receptor response. The EGFR specifically activates phospholipase C-gamma1 (PLC-gamma1). Recruitment of substrate molecules with Src homology 2 (SH2) domains or phosphotyrosine binding (PTB) domains to phosphotyrosines in the receptor is one of the factors creating substrate specificity. In addition, it has been shown that the TK domains of the IR and EGFR show preferences to phosphorylate distinct peptides in vitro, suggesting additional mechanisms of substrate recognition. We have examined to what extent the substrate preference of the TK domain contributes to the specificity of the receptor in vivo. For this purpose we determined whether the IR TK domain, in situ, is able to tyrosine-phosphorylate substrates normally used by the EGFR. A chimaeric receptor, consisting of an EGFR in which the juxtamembrane and tyrosine kinase domains were exchanged by their IR counterparts, was expressed in CHO-09 cells lacking endogenous EGFR. This receptor was found to activate PLC-gamma1, indicating that the IR TK domain, in situ, is able to tyrosine phosphorylate substrates normally used by the EGFR. These findings suggest that the IR TK domain, in situ, has a low specificity for selection and phosphorylation of non-cognate substrates.