New article published in PLos One

Mária Pukáncsik and Kinga Nagy article is published in PLos One.

Hereby you may find some highlights making you eager to read this very interesting article.

A novel uracil-DNA degrading protein factor (termed UDE) was identified in Drosophila melanogaster with no significant structural and functional homology to other uracil-DNA binding or processing factors. Determination of the 3D structure of UDE is excepted to provide key information on the description of the molecular mechanism of action of UDE catalysis, as well as in general uracil-recognition and nuclease action. Towards this long-term aim, the random library ESPRIT technology was applied to the novel protein UDE to overcome problems in identifying soluble expressing constructs given the absence of precise information on domain content and arrangement. Nine constructs of UDE were chosen to decipher structural and functional relationships. Vacuum ultraviolet circular dichroism (VUVCD) spectroscopy was performed to define the secondary structure content and location within UDE and its truncated variants. The quantitative analysis demonstrated exclusive α-helical content for the full-length protein, which is preserved in the truncated constructs. Arrangement of α-helical bundles within the truncated protein segments suggested new domain boundaries which differ from the conserved motifs determined by sequence-based alignment of UDE homologues. Here we demonstrate that the combination of ESPRIT and VUVCD spectroscopy provides a new structural description of UDE and confirms that the truncated constructs are useful for further detailed functional studies.

nagy kinga cikk jo

A) Secondary structure content determination of UDE and its fragments using VUVCD and SELCON3 program. Vacuum-ultraviolet circular dichroism (Δε) spectra of the UDE protein and some of its truncated fragments measured over the wavelength region of λ=170-255 nm. spectrum of UDE is shown in red. B) Final estimate for the secondary structure of UDE obtained as an average of the structure of full-length UDE protein and in its nine truncated fragments were determined from the CD spectra and the amino acid sequence using a neural network algorithm. The α-helical segments and β-strands are displayed in blue and red, respectively, while both turns and disordered parts appear in yellow.

Article of Judit Szabó et al. published in Scientific Reports

Article of Judit Szabó et al. is published in Scientific Reports      
(http://www.nature.com/articles/srep24219).                  
Scientific Reports

Hereby you may find some highlights making you eager to read this very interesting article.

Abstract
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 third member of the superfamily, has been a question of debate for decades. Therefore, we designed hybrid dUTPase trimers to access conformational states potentially mimicking the ones observed in the cooperative relatives. We studied how the interruption of different steps of the enzyme cycle affects the active site cross talk. We found that subunits work independently in dUTPase. The experimental results combined with a comparative structural analysis of dUTPase superfamily enzymes revealed that subtile structural differences within the allosteric loop and the central channel in these enzymes give rise to their dramatically different cooperative behavior. We demonstrate that the lack of allosteric regulation in dUTPase is related to the functional adaptation to more efficient dUTP hydrolysis which is advantageous in uracil-DNA prevention.

Asymmetric hybrid enzymes exhibit non-cooperative kinetics in the different reaction steps.

SZJ SciRep Fig

(a) Schematic representation of the created hybrids (covalent heterotrimers). Blue, red and yellow spheres represent dUTPase protomers containing the D102N, A98F and T148STOP mutations, respectively. Note, that all protomers contain the F158W mutation as well, except for the T148STOP mutant. Grey areas indicate enzymatic inactivity. (b) Steady-state kinetics of human dUTPase constructs: WWW (solid square), WWN (solid triangles), WNN (open triangles), WWF (solid stars), WWS (solid circle). Smooth lines through the data are hyperbolic fits yielding Vmax= 7.9 ± 0.5 s−1 for WWW, Vmax = 4.4 ± 0.2 s−1 for WWF, Vmax = 3.8 ± 0.2 s−1 for WWN, Vmax = 1.8 ± 0.1 s−1 for WNN, Vmax = 2.9 ± 0.3 s−1 for WWS. KM values are listed in Table 1. (c) Comparison of the catalytic constants (striped bar) and apparent catalytic constants (grey bar) for determined by single turnover (transient kinetics) and steady-state experiments, respectively. See also Table 1 for the data. (d) Fluorescence intensity titrations upon dUTP binding to the various dUTPase constructs measured by stopped-flow (the symbol code is identical to that in panel (b). Smooth lines through data are hyperbolic fits yielding Kd values summarized in Table 1.

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