Judit Tóth

Reserach Centre for Natural Sciences, Hungarian Academy of Sciences · Institute of Enzymology

The concerted action of DNA replication and cell division has been extensively investigated in eukaryotes. Well demarcated checkpoints have been identified in the cell cycle, which provides the correct DNA stoichiometry and appropriate growth in the progeny. In bacteria, which grow faster and less concerted than eukaryotes, the linkages between cel...

Stimulated by the growing interest in the role of dNTP pools in physiological and malignant processes, we established dNTPpoolDB, the database that offers access to quantitative data on dNTP pools from a wide range of species, experimental and developmental conditions (https://dntppool.org/). The database includes measured absolute or relative cell...

Pathogenicity islands ofStaphylococcus aureusare under the strong control of helper phages, where regulation is communicated at the gene expression level via a family of specific repressor proteins. The repressor proteins are crucial to phage-host interactions and, based on their protein characteristics, may also be exploited as versatile molecular...

dUTPase superfamily enzymes generate dUMP, the obligate precursor for de novo dTTP biosynthesis, from either dUTP (monofunctional dUTPase, Dut) or dCTP (bifunctional dCTP deaminase/dUTPase, Dcd:dut). In addition, the elimination of dUTP by these enzymes prevents harmful uracil incorporation into DNA. These two beneficial outcomes have been thought...

dUTPases catalyze the hydrolysis of dUTP into dUMP and pyrophosphate to maintain the proper nucleotide pool for DNA metabolism. Recent evidence suggests dUTPases may also represent a selective drug target in mycobacteria because of these enzymes' crucial role in maintaining DNA integrity. Nucleotide-hydrolyzing enzymes typically harbor a buried lig...

Arginine finger is a highly conserved and essential residue in many GTPase and AAA+ ATPase enzymes that completes the active site from a distinct protomer, forming contacts with the γ-phosphate of the nucleotide. To date, no pyrophosphatase has been identified that employs an arginine finger fulfilling all the above properties, all essential argini...

Members of the dUTPase superfamily play an important role in the maintenance of the pyrimidine nucleotide balance and of genome integrity. dCTP deaminases and the bifunctional dCTP deaminase-dUTPases are cooperatively regulated by dTTP. However, the manifestation of allosteric behavior within the same trimeric protein architecture of dUTPases, the...

Most enzymes present a catalytic mechanism where one or more proton transfer events occur coupled tightly together with the enzymatic chemical reaction. We show here that inactivating mutations decouple this proton transfer step from the phosphate cleavage reaction in dUTPase. Homotrimeric dUTPase enzymes catalyze the hydrolysis of dUTP to dUMP and...

Proteins responsible for the integrity of the genome are often used targets in drug therapies against various diseases. The inhibitors of these proteins are also important to study the pathways in genome integrity maintenance. A prominent example is Ugi, a well known cross-species inhibitor protein of the enzyme uracil-DNA glycosylase, responsible...

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expressi...

Abstract Phosphorylation by the cyclin-dependent kinase 1 (Cdk1) adjacent to nuclear localization segments (NLSs) is an important mechanism of regulation of nucleocytoplasmic transport. However, no systematic survey has yet been performed in human cells to analyze this regulatory process, and the corresponding cell-cycle dynamics have not yet been...

Genome integrity requires well controlled cellular pools of nucleotides. dUTPases are responsible for regulating cellular dUTP levels and providing dUMP for dTTP biosynthesis. In Staphylococcus , phage dUTPases are also suggested to be involved in a moonlighting function regulating the expression of pathogenicity-island genes. Staphylococcal phage...

Phosphorylation adjacent to nuclear localization signals (NLSs) is involved in the regulation of nucleocytoplasmic transport. The nuclear isoform of human dUTPase, an enzyme that is essential for genomic integrity, has been shown to be phosphorylated on a serine residue (Ser11) in the vicinity of its nuclear localization signal; however, the effect...

Thymidine biosynthesis is essential in all cells. Inhibitors of the enzymes involved in this pathway (e.g. methotrexate) are thus frequently used as cytostatics. Due to its pivotal role in mycobacterial thymidylate synthesis dUTPase, which hydrolyzes dUTP into the dTTP precursor dUMP, has been suggested as a target for new antitubercular agents. Al...

Primers used in the present study. (DOCX)

Key plasmids used in the generation of dut deletion mutant M. smegmatis. (A) p2Nbk-duth delivery vector used to generate mutant SCOs. The 2.1 kb HindIII M. smegmatis fragment indicated in Figure 3 was inserted into the p2NIL vector to construct the delivery vector. The dut allele was disrupted with a 1.8 kb fragment encoding hygromycin resistance,...

We investigated the potential (d)NDP/(d)NTP discrimination mechanisms in nucleotide pyrophosphatases. Here, we report that dUTPase, an essential nucleotide pyrophosphatase, uses a C-terminal P-loop-like sequence in a unique mechanism for substrate discrimination and efficient hydrolysis. Our spectroscopy and transient kinetics results on human dUTP...

Proteins of each cluster. List of the 3,948 MTB proteins. Proteins are defined with Uniprot IDs. For each protein, the number of the containing cluster is given. (XLS)

PE/PPE family proteins. List of proteins belonging to the PE/PPE protein family. Proteins are defined with Uniprot IDs. For each protein, the pknX name is given. (XLS)

MTB protein profiles. Profiles for each of the 3,948 MTB proteins obtained from the alignments performed on the CPSD (see Methods). (XLS)

pknA protein predictions. Structure predictions for the pknA protein, including IUPred, ANCHOR and PSIPRED. In the top part the kinase domain is shown in green, the transmembrane region is shown in black and disordered regions predicted by IUPred are shown in red. Solid black and striped boxes in PSIPRED predictions indicate predicted α helixes and...

pkn proteins. List of proteins belonging to the pkn protein family. Proteins are defined with Uniprot IDs. For each protein, the pknX name is given. (XLS)

Proteins of mycolic acid synthesis. List of proteins involved in the synthesis of mycolic acid. The corresponding functions are shown together with the name and position of the domains present. The last 5 columns show the number of occurrences of the respective domains in various groups of organisms. Bacterial specific domains are highlighted. (XLS...

Lengthy co-evolution of Homo sapiens and Mycobacterium tuberculosis, the main causative agent of tuberculosis, resulted in a dramatically successful pathogen species that presents considerable challenge for modern medicine. The continuous and ever increasing appearance of multi-drug resistant mycobacteria necessitates the identification of novel dr...

dUTPase is involved in preserving DNA integrity in cells. We report an efficient dUTPase silencing by RNAi-based system in stable human cell line. Repression of dUTPase induced specific expression level increments for thymidylate kinase and thymidine kinase, and also an increased sensitization to 5-fluoro-2'-deoxyuridine and 5-fluoro-uracil. The ca...

Autophagy is a lysosome-mediated self-degradation process of eukaryotic cells that, depending on the cellular milieu, can either promote survival or act as an alternative mechanism of programmed cell death (PCD) in terminally differentiated cells. Despite the important developmental and medical implications of autophagy and the main form of PCD, ap...

Aromatic interactions are well-known players in molecular recognition but their catalytic role in biological systems is less documented. Here, we report that a conserved aromatic stacking interaction between dUTPase and its nucleotide substrate largely contributes to the stabilization of the associative type transition state of the nucleotide hydro...

Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylch...

dUTP pyrophosphatases (dUTPases) are essential for genome integrity. Recent results allowed characterization of the role of conserved residues. Here we analyzed the Asp/Asn mutation within conserved Motif I of human and mycobacterial dUTPases, wherein the Asp residue was previously implicated in Mg(2+)-coordination. Our results on transient/steady-...

Lipid-protein interactions drive core cellular signaling events and regulate the function of most membrane proteins. Despite their significance, molecular details of these interactions are scarcely known. Here we provide mechanistic insight into the binding of the lipid mediator sphingosylphosphorylcholine to calmodulin, the most central and ubiqui...

The nuclear isoform of human dUTPase plays an important role in maintaining genomic integrity. Its expression is strictly cell cycle regulated and is known to be a phosphoprotein in vivo. However, the role of this phosphorylation remained unknown.Here we show regulation of the nuclear transport of human dUTPase via phosphorylation of a serine resid...

The phosphate-binding loop (P-loop) or Walker A sequence is a common feature of a large number of ATP and GTP binding proteins including kinases, cytoskeleton and DNA motors, membrane pumps and transporters. All known P-loop containing proteins are able to sense the difference between bound NTP and NDP via their P-loop which allows for hydrolyzing...

The dUTPase enzymatic activity is indispensable to efficiently reduce cellular dUTP/dTTP levels. Lack of the enzyme leads to erroneous uracil incorporation into DNA resulting in chromosome fragmentation and cell death. dUTPase is therefore reported to be a preventive DNA repair factor and a high-potential drug target in cancer. Although divalent me...

Previously we have identified the lipid mediator sphingosylphosphorylcholine (SPC) as the first potentially endogenous inhibitor of the ubiquitous Ca2+ sensor calmodulin (CaM) (Kovacs, E., and Liliom, K. (2008) Biochem. J. 410, 427–437). Here we give mechanistic insight into CaM inhibition by SPC, based on fluorescence stopped-flow studies with the...

Previously we have identified the lipid mediator sphingosylphosphorylcholine (SPC) as the first potentially endogenous inhibitor of the ubiquitous Ca2+ sensor calmodulin (CaM) (Kovacs, E., and Liliom, K. (2008) Biochem. J. 410, 427-437). Here we give mechanistic insight into CaM inhibition by SPC, based on fluorescence stopped-flow studies with the...

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We have determined the kinetic mechanism and motile properties of the switch 1 mutant S217A of myosin Va. Phosphate dissociation from myosin V-ADP-Pi (inorganic phosphate) and actomyosin V-ADP-Pi and the rate of the hydrolysis step (myosin V-ATP-->myosin V-ADP-Pi) were all approximately 10-fold slower in the S217A mutant than in wild type (WT) myos...

The thymine−uracil exchange constitutes one of the major chemical differences between DNA and RNA. Although these two bases form the same Watson−Crick base pairs with adenine and are equivalent for both information storage and transmission, uracil incorporation in DNA is usually a mistake that needs to be excised. There are two ways for uracil to a...

Human dUTPase is essential in controlling relative cellular levels of dTTP/dUTP, both of which can be incorporated into DNA. The nuclear isoform of the enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies. The recently determined three-dimensional structure of this protein in complex with an isosteric subs...

Human dUTPase, essential for DNA integrity, is an important survival factor for cancer cells. We determined the crystal structure of the enzyme:alpha,beta-imino-dUTP:Mg complex and performed equilibrium binding experiments in solution. Ordering of the C-terminus upon the active site induces close juxtaposition of the incoming nucleophile attacker w...

After ATP binding the myosin head undergoes a large structural rearrangement called the recovery stroke. This transition brings catalytic residues into place to enable ATP hydrolysis, and at the same time it causes a swing of the myosin lever arm into a primed state, which is a prerequisite for the power stroke. By introducing point mutations into...

Little is known about the functions of class III unconventional myosins although, with an N-terminal kinase domain, they are potentially both signaling and motor proteins. Limulus myosin III is particularly interesting because it is a phosphoprotein abundant in photoreceptors that becomes more heavily phosphorylated at night by protein kinase A. Th...

(–)-Blebbistatin (1), a recently discovered small molecule inhibitor of the ATPase activity of non-muscle myosin II has been prepared from methyl 5-methylanthranilate (6) in three steps. This flexible synthetic route has also been used to prepare a nitro group-containing analogue 12 that has modified fluorescence properties and improved stability u...

Myosin V is the best characterized vesicle transporter in vertebrates, but it has been unknown as to whether all members of the myosin V family share a common, evolutionarily conserved mechanism of action. Here we show that myosin V from Drosophila has a strikingly different motor mechanism from that of vertebrate myosin Va, and it is a nonprocessi...

Blebbistatin is a recently discovered small molecule inhibitor showing high affinity and selectivity toward myosin II. Here we report a detailed investigation of its mechanism of inhibition. Blebbistatin does not compete with nucleotide binding to the skeletal muscle myosin subfragment-1. The inhibitor preferentially binds to the ATPase intermediat...

The enzymatic and motor function of smooth muscle and nonmuscle myosin II is activated by phosphorylation of the regulatory light chains located in the head portion of myosin. Dimerization of the heads, which is brought about by the coiled-coil tail region, is essential for regulation since single-headed fragments are active regardless of the state...

Lys84 of skeletal muscle myosin located at the interface between the motor and neck domains has long been utilized as a useful chemical probe sensing motor domain conformational changes and tilting of the lever arm. Here we report the first site-directed mutagenesis study on this side chain and its immediate chemical environment. We made Dictyostel...

A pályázat során a miozin motorfehérjék szerkezet-funkció összefüggéseit vizsgáltuk. A pályázat talán legfontosabb eredménye, hogy elsőnek sikerült atomi felbontású képet kapnunk a miozin coiled-coil (alfa-helikális dimer) szerkezetű farok régió egy részletéről, ráadásul egy olyan instabil doménről, amely a konvencionális miozin (II-es típus) működ...