20 research outputs found
Crystallization and preliminary crystallographic analysis of dUTPase from the helper phage Φ11 of Staphylococcus aureus
Staphylococcus aureus superantigen-carrying pathogenicity islands (SaPIs) have a
determinant role in spreading virulence genes among bacterial populations that constitute a
major health hazard. Repressor (Stl) proteins are responsible for transcriptional regulation of
pathogenicity island genes. Recently, a derepressing interaction between the repressor Stl
SaPIbov1 with dUTPase from the Φ11 helper phage was suggested [Tormo-Mas et al. (2010).
Nature 465, 779-782]. Towards elucidating the molecular mechanism of this interaction, this
study reports expression, purification, and X-ray analysis of Φ11 dUTPase that contains a
phage-specific polypeptide segment not present in other dUTPases. Crystals were obtained
using the hanging-drop vapor-diffusion method at room temperature. Data were collected
from one type of crystal to 2.98 Å resolution. The crystal of Φ11 dUTPase belonged to the
cubic space group I23, with unit-cell parameters a=98.16 Å, α=β=γ= 90.00o
Cellular response to efficient dUTPase RNAi silencing in stable HeLa cell lines perturbs expression levels of genes involved in thymidylate metabolism.
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 catalytic mechanism of dUTPase was investigated for 5-fluoro-dUTP. The 5F-substitution on the uracil ring of the substrate did not change the kinetic mechanism of dUTP hydrolysis by dUTPase. Results indicate that RNAi silencing of dUTPase induces a complex cellular response wherein sensitivity towards fluoropyrimidines and gene expression levels of related enzymes are both modulated
Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus
The sequence of a phenylalanine ammonia-lyase (PAL; EC: 4.3.1.24) of the thermophilic and radiotolerant bacterium
Rubrobacter xylanophilus (RxPAL) was identified by screening the genomes of bacteria for members of the phenylalanine
ammonia-lyase family. A synthetic gene encoding the RxPAL protein was cloned and overexpressed in Escherichia coli TOP
10 in a soluble form with an N-terminal His6-tag and the recombinant RxPAL protein was purified by Ni-NTA affinity
chromatography. The activity assay of RxPAL with L-phenylalanine at various pH values exhibited a local maximum at pH 8.5
and a global maximum at pH 11.5. Circular dichroism (CD) studies showed that RxPAL is associated with an extensive ahelical
character (far UV CD) and two distinctive near-UV CD peaks. These structural characteristics were well preserved up
to pH 11.0. The extremely high pH optimum of RxPAL can be rationalized by a three-dimensional homology model
indicating possible disulfide bridges, extensive salt-bridge formation and an excess of negative electrostatic potential on the
surface. Due to these properties, RxPAL may be a candidate as biocatalyst in synthetic biotransformations leading to
unnatural L- or D-amino acids or as therapeutic enzyme in treatment of phenylketonuria or leukemia
Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control
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 expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements
Catalytic mechanism of alpha-phosphate attack in dUTPase is revealed by X-ray crystallographic snapshots of distinct intermediates, 31P-NMR spectroscopy and reaction path modelling.
Enzymatic synthesis and hydrolysis of nucleoside phosphate compounds play a key role in various biological pathways, like signal transduction, DNA synthesis and metabolism. Although these processes have been studied extensively, numerous key issues regarding the chemical pathway and atomic movements remain open for many enzymatic reactions. Here, using the Mason-Pfizer monkey retrovirus dUTPase, we study the dUTPase-catalyzed hydrolysis of dUTP, an incorrect DNA building block, to elaborate the mechanistic details at high resolution. Combining mass spectrometry analysis of the dUTPase-catalyzed reaction carried out in and quantum mechanics/molecular mechanics (QM/MM) simulation, we show that the nucleophilic attack occurs at the alpha-phosphate site. Phosphorus-31 NMR spectroscopy (31P-NMR) analysis confirms the site of attack and shows the capability of dUTPase to cleave the dUTP analogue alpha,beta-imido-dUTP, containing the imido linkage usually regarded to be non-hydrolyzable. We present numerous X-ray crystal structures of distinct dUTPase and nucleoside phosphate complexes, which report on the progress of the chemical reaction along the reaction coordinate. The presently used combination of diverse structural methods reveals details of the nucleophilic attack and identifies a novel enzyme-product complex structure
Structure and enzymatic mechanism of a moonlighting dUTPase
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 trimeric dUTPase sequences include a specific insertion that is not found in other organisms. Here, a 2.1 Å resolution three-dimensional structure of a [varphi]11 phage dUTPase trimer with complete localization of the phage-specific insert, which folds into a small [beta]-pleated mini-domain reaching out from the dUTPase core surface, is presented. The insert mini-domains jointly coordinate a single Mg2+ ion per trimer at the entrance to the threefold inner channel. Structural results provide an explanation for the role of Asp95, which is suggested to have functional significance in the moonlighting activity, as the metal-ion-coordinating moiety potentially involved in correct positioning of the insert. Enzyme-kinetics studies of wild-type and mutant constructs show that the insert has no major role in dUTP binding or cleavage and provide a description of the elementary steps (fast binding of substrate and release of products). In conclusion, the structural and kinetic data allow insights into both the phage-specific characteristics and the generally conserved traits of [varphi]11 phage dUTPase
The new truffle genus Babosia and a new species of Stouffera from semiarid grasslands of Hungary
Truffles with distinct morphological and anatomical features were collected during a study of
hypogeous fungi of semiarid sandy grasslands of the Great Hungarian Plain in Hungary, representing
the westernmost localities of the Eurasian steppe belt. None of the ascomata were collected
near ectomycorrhizal plant species, and none were identified as ectomycorrhizal during previous
surveys in the collection area. We studied morphoanatomical characteristics of these truffles with
light and scanning electron microscopy and investigated their phylogenetic positions based on
analyses of different nuclear loci. The truffles were found to represent two novel lineages that
grouped with the Marcelleina-Peziza gerardii clade of the Pezizaceae. One formed a distinct lineage,
for which we propose a new genus Babosia with a new species Babosia variospora characterized by
diverse spore ornamentation varying even within one ascus. The truffles in the other lineage
clustered with the rarely collected American truffle Stouffera longii and share with it similar spore
ornamentation and habitat features. However, our material differs from S. longii by geographic
origin, the quick and strong coloration of the ascomata to dark gray at cut surface or bruised area,
varying spore number in asci, and smaller spore size; thus, we describe it as a new species, Stouffera
gilkeyae
Inhabiting plant roots, nematodes, and truffles— Polyphilus , a new helotialean genus with two globally distributed species
International audienceFungal root endophytes, including the common group of dark septate endophytes (DSEs), represent different taxonomic groups and potentially diverse life strategies. In this study, we investigated two unidentified helotialean lineages found previously in a study of DSE fungi of semiarid grasslands, from several other sites, and collected recently from a pezizalean truffle ascoma and eggs of the cereal cyst nematode Heterodera filipjevi. The taxonomic positions and phylogenetic relationships of 21 isolates with different hosts and geographic origins were studied in detail. Four loci, namely, nuc rDNA ITS1-5.8S-ITS2 (internal transcribed spacer [ITS]), partial 28S nuc rDNA (28S), partial 18S nuc rDNA (18S), and partial RNA polymerase II second-largest subunit (RPB2), were amplified and sequenced for molecular phylogenetic analyses. Analyses of similar ITS sequences from public databases revealed two globally distributed lineages detected in several biomes from different geographic regions. The host interaction of isolates from nematodes was examined using in vitro bioassays, which revealed that the fungi could penetrate nematode cysts and colonize eggs of H. filipjevi, confirming observations from field-collected samples. This is the first report of a DSE, and we are not aware of other helotialean fungal species colonizing the eggs of a plant-parasitic nematode. Neither conidiomata and conidia nor ascomata formation was detected in any of the isolates. Based on molecular phylogenetic analyses, these isolates represent a distinct lineage within the Helotiales in the Hyaloscyphaceae. For this lineage, we propose here the new genus Polyphilus represented by two new species, P. sieberi and P. frankenii
NLS copy number variation governs efficiency of nuclear import: case study on dUTPases
Nucleocytoplasmic trafficking of large macromolecules requires an active
transport machinery. In many cases, this is initiated by binding of the
nuclear localization signal (NLS) peptide of cargo proteins to importin-a
molecules. Fine orchestration of nucleocytoplasmic trafficking is of particularly
high importance for proteins involved in maintenance of genome
integrity, such as dUTPases, which are responsible for prevention of uracil
incorporation into the genome. In most eukaryotes, dUTPases have two
homotrimeric isoforms: one of these contains three NLSs and is present in
the cell nucleus, while the other is located in the cytoplasm or the mitochondria.
Here we focus on the unusual occurrence of a pseudo-heterotrimeric
dUTPase in Drosophila virilis that contains one NLS, and investigate
its localization pattern compared to the homotrimeric dUTPase isoforms
of Drosophila melanogaster. Although the interaction of individual NLSs
with importin-a has been well characterized, the question of how multiple
NLSs of oligomeric cargo proteins affect their trafficking has been less frequently
addressed in adequate detail. Using the D. virilis dUTPase as a
fully relevant physiologically occurring model protein, we show that NLS
copy number influences the efficiency of nuclear import in both insect and
mammalian cell lines, as well as in D. melanogaster and D. virilis tissues.
Biophysical data indicate that NLS copy number determines the stoichiometry
of complexation between importin-a and dUTPases. The main conclusion
of our study is that, in D. virilis, a single dUTPase isoform efficiently
reproduces the cellular dUTPase distribution pattern that requires two isoforms
in D. melanogaster
Structural properties of <i>Rx</i>PAL by homology modeling.
<p>Ribbon representation of the homotetrameric model of <i>Rx</i>PAL (Panel A) and backbone line representation model with the MIO (in pink) surrounded by three pairs of cysteines (in yellow) which may form two intra-subunit and one inter-subunit disulfide bridges (Panel B). The electrostatic surface potential representation of <i>Rx</i>PAL (Panel C) compared to that of less alkalophilic <i>Av</i>PAL (Panel D, PDB code 3CZO <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085943#pone.0085943-Wang1" target="_blank">[35]</a>) indicated highly negatively charged <i>Rx</i>PAL surface.</p