Crystal Structure of 4-Chlorocatechol 1,2-Dioxygenase from the Chlorophenol-utilizing Gram-positive Rhodococcus opacus 1CP

The crystal structure of the 4-chlorocatechol 1,2-dioxygenase from the Gram-positive bacterium Rhodococcus opacus (erythropolis) 1CP, a Fe(III) ion-containing enzyme involved in the aerobic biodegradation of chloroaromatic compounds, has been solved by multiple wavelength anomalous dispersion using the weak anomalous signal of the two catalytic irons (1 Fe/257 amino acids) and refined at a 2.5 A resolution (R(free) 28.7%; R factor 21.4%). The analysis of the structure and its comparison with the structure of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus ADP1 (Ac 1,2-CTD) highlight significant differences between these enzymes. The general topology of the present enzyme comprises two catalytic domains (one for each subunit) related by a noncrystallographic 2-fold axis and separated by a common alpha-helical zipper motif consisting of five N-terminal helices from each subunit; furthermore the C-terminal tail is shortened significantly with respect to the known Ac 1,2-CTD. The presence of two phospholipids binding in a hydrophobic tunnel along the dimer axis is shown here to be a common feature for this class of enzyme. The active site cavity presents several dissimilarities with respect to the known catechol-cleaving enzyme. The catalytic nonheme iron(III) ion is bound to the side chains of Tyr-134, Tyr-169, His-194, and His-196, and a cocrystallized benzoate ion, bound to the metal center, reveals details on a novel mode of binding of bidentate inhibitors and a distinctive hydrogen bond network with the surrounding ligands. Among the amino acid residues expected to interact with substrates, several are different from the corresponding analogs of Ac 1,2-CTD: Asp-52, Ala-53, Gly-76, Phe-78, and Cys-224; in addition, regions of largely conserved amino acid residues in the catalytic cleft show different shapes resulting from several substantial backbone and side chain shifts. The present structure is the first of intradiol dioxygenases that specifically catalyze the cleavage of chlorocatechols, key intermediates in the aerobic catabolism of toxic chloroaromatics

Employing infrared microscopy (IRM) in combination with a pre-trained neural network to visualise and analyse the defect distribution in Cadmium Telluride crystals

While Cadmium Telluride (CdTe) excels in terms of photon radiation absorption properties and outperforms silicon (Si) in this respect, the crystal growth, characterization and processing into a radiation detector is much more complicated. Additionally, large concentrations of extended crystallographic defects, such as grain boundaries, twins, and tellurium (Te) inclusions, vary from crystal to crystal and can reduce the spectroscopic performance of the processed detector. A quality assessment of the material prior to the complex fabrication process is therefore crucial. To locate the Te-defects, we scan the crystals with infrared microscopy (IRM) in different layers, obtaining a 3D view of the defect distribution. This provides us with important information on the defect density and locations of Te inclusions, and thus a handle to assess the quality of the material. For the classification of defects in the large amount of IRM image data, a convolutional neural network is employed. From the post-processed and analysed IRM data, 3D defect maps of the CdTe crystals are created, which make different patterns of defect agglomerations inside the crystals visible. In total, more than 100 crystals were scanned with the current IRM setup. In this paper, we compare two crystal batches, each consisting of 12 samples. We find significant differences in the defect distributions of the crystals.Peer reviewe

Crystal structure of a blue laccase from Lentinus tigrinus: evidences for intermediates in the molecular oxygen reductive splitting by multicopper oxidases

<p>Abstract</p> <p>Background</p> <p>Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in pathogenesis, immunogenesis and morphogenesis of organisms and in the metabolic turnover of complex organic substances. They catalyze the coupling between the four one-electron oxidations of a broad range of substrates with the four-electron reduction of dioxygen to water. These catalytic processes are made possible by the contemporaneous presence of at least four copper ion sites, classified according to their spectroscopic properties: one type 1 (T1) site where the electrons from the reducing substrates are accepted, one type 2 (T2), and a coupled binuclear type 3 pair (T3) which are assembled in a T2/T3 trinuclear cluster where the electrons are transferred to perform the O₂reduction to H₂O.</p> <p>Results</p> <p>The structure of a laccase from the white-rot fungus <it>Lentinus (Panus) tigrinus</it>, a glycoenzyme involved in lignin biodegradation, was solved at 1.5 Å. It reveals a asymmetric unit containing two laccase molecules (A and B). The progressive reduction of the copper ions centers obtained by the long-term exposure of the crystals to the high-intensity X-ray synchrotron beam radiation under aerobic conditions and high pH allowed us to detect two sequential intermediates in the molecular oxygen reduction pathway: the "peroxide" and the "native" intermediates, previously hypothesized through spectroscopic, kinetic and molecular mechanics studies. Specifically the electron-density maps revealed the presence of an end-on bridging, μ-η₁:η₁peroxide ion between the two T3 coppers in molecule B, result of a two-electrons reduction, whereas in molecule A an oxo ion bridging the three coppers of the T2/T3 cluster (μ3-oxo bridge) together with an hydroxide ion externally bridging the two T3 copper ions, products of the four-electrons reduction of molecular oxygen, were best modelled.</p> <p>Conclusion</p> <p>This is the first structure of a multicopper oxidase which allowed the detection of two intermediates in the molecular oxygen reduction and splitting. The observed features allow to positively substantiate an accurate mechanism of dioxygen reduction catalyzed by multicopper oxidases providing general insights into the reductive cleavage of the O-O bonds, a leading problem in many areas of biology.</p

Modeling the impact of defects on the charge collection efficiency of a Cadmium Telluride detector

Cadmium telluride is a favorable material for X-ray detection as it has an outstanding characteristic for room temperature operation. It is a high-Z material with excellent photon radiation absorption properties. However, CdTe single crystals may include a large number of extended crystallographic defects, such as grain boundaries, twins, and tellurium (Te) inclusions, which can have an impact on detector performance. A Technology Computer Aided Design (TCAD) local defect model has been developed to investigate the effects of local defects on charge collection efficiency (CCE). We studied a 1 mm thick Schottky-type CdTe radiation detector with transient current technique by using a red laser at room temperature. By raster scanning the detector surface we were able to study signal shaping within the bulk, and to locate surface defects by observing their impact on the CCE. In this paper we present our TCAD model with localized defect, and compare the simulation results to TCT measurements. In the model an inclusion with a diameter of 10 mu m was assumed. The center of the defect was positioned at 6 mu m distance from the surface. We show that the defect has a notable effect on current transients, which in turn affect the CCE of the CdTe detector. The simulated charge collection at the position of the defect decreases by 80 % in comparison to the defect-free case. The simulations show that the defects give a characteristic shape to TCT signal. This can further be used to detect defects in CdTe detectors and to estimate the overall defect density in the material.Peer reviewe

Crystal Structure of the Hydroxyquinol 1,2-Dioxygenase from Nocardioides simplex 3E, a Key Enzyme Involved in Polychlorinated Aromatics Biodegradation

Hydroxyquinol 1,2-dioxygenase (1,2-HQD) catalyzes the ring cleavage of hydroxyquinol (1,2,4-trihydroxybenzene), a central intermediate in the degradation of aromatic compounds including a variety of particularly recalcitrant polychloro- and nitroaromatic pollutants. We report here the primary sequence determination and the analysis of the crystal structure of the 1,2-HQD from Nocardioides simplex 3E solved at 1.75 A resolution using the multiple wavelength anomalous dispersion of the two catalytic irons (1 Fe/293 amino acids). The catalytic Fe(III) coordination polyhedron composed by the side chains of Tyr164, Tyr197, His221, and His223 resembles that of the other known intradiol-cleaving dioxygenases, but several of the tertiary structure features are notably different. One of the most distinctive characteristics of the present structure is the extensive openings and consequent exposure to solvent of the upper part of the catalytic cavity arranged to favor the binding of hydroxyquinols but not catechols. A co-crystallized benzoate-like molecule is also found bound to the metal center forming a distinctive hydrogen bond network as observed previously also in 4-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus 1CP. This is the first structure of an intradiol dioxygenase specialized in hydroxyquinol ring cleavage to be investigated in detail

Quality assessment of cadmium telluride as a detector material for multispectral medical imaging

Cadmiumtelluride (CdTe) is a high-Z material with excellent photon radiation absorption properties, making it a promising material to include in radiation detection technologies. However, the brittleness of CdTe crystals as well as their varying concentration of defects necessitate a thorough quality assessment before the complex detector processing procedure. We present our quality assessment of CdTe as a detector material for multispectralmedical imaging, a research which is conducted as part of the Consortium Project Multispectral Photon-counting for Medical Imaging and Beam characterization (MPMIB). The aim of the project is to develop novel CdTe detectors and obtain spectrum-per-pixel information that make the distinction between different radiation types and tissues possible. To evaluate the defect density inside the crystals - which can deteriorate the detector performance - we employ infrared microscopy (IRM). Posterior data analysis allows us to visualise the defect distributions as 3D defect maps. Additionally, we investigate front and backside differences of the material with current-voltage (IV) measurements to determine the preferred surface for the pixelisation of the crystal, and perform test measurements with the prototypes to provide feedback for further processing. We present the different parts of our quality assessment chain and will close with first experimental results obtained with one of our prototype photon-counting detectors in a small tomographic setup.Peer reviewe

Characterization of magnetic Czochralski silicon devices with aluminium oxide field insulator : effect of oxygen precursor on electrical properties and radiation hardness

Aluminium oxide (Al2O3) has been proposed as an alternative to thermal silicon dioxide (SiO2) as field insulator and surface passivation for silicon detectors, where it could substitute p-stop/p-spray insulation implants between pixels due to its negative oxide charge, and enable capacitive coupling of segments by means of its higher dielectric constant. Al2O3 is commonly grown by atomic layer deposition (ALD), which allows the deposition of thin layers with excellent precision. In this work, we report the electrical characterization of single pad detectors (diodes) and MOS capacitors fabricated on magnetic Czochralski silicon substrates and using Al2O3 as field insulator. Devices are studied by capacitance-voltage, current-voltage, and transient current technique measurements. We evaluate the influence of the oxygen precursors in the ALD process, as well as the effect of gamma irradiation, on the properties of these devices. We observe that leakage currents in diodes before the onset of breakdown are low for all studied ALD processes. Charge collection as measured by transient current technique (TCT) is also independent of the choice of oxygen precursor. The Al2O3 films deposited with O-3 possess a higher negative oxide charge than films deposited by H2O, However, in diodes a higher oxide charge is linked to earlier breakdown, as has been predicted by simulation studies. A combination of H2O and O-3 precursors results in a good compromise between the beneficial properties provided by the respective individual precursors.Peer reviewe

Processing of AC-coupled n-in-p pixel detectors on MCz silicon using atomic layer deposited aluminium oxide

We report on the fabrication of capacitively (AC) coupled n(+)-in-p pixel detectors on magnetic Czochralski silicon substrates. In our devices, we employ a layer of aluminium oxide (Al2O3) grown by atomic layer deposition (ALD) as dielectric and field insulator, instead of the commonly used silicon dioxide (SiO2). As shown in earlier research, Al2O3 thin films exhibit high negative oxide charge, and can thus serve as a substitute for p-stop/p-spray insulation implants between pixels. In addition, they provide far higher capacitance densities than SiO2 due to their high dielectric constant, permitting more efficient capacitive coupling of pixels. Furthermore, metallic titanium nitride (TiN) bias resistors are presented as an alternative to punch-through or poly-Si resistors. Devices obtained by the above mentioned process are characterized by capacitance-voltage and current-voltage measurements, and by 2 MeV proton microprobe. Results show the expected high negative charge of the Al2O3 dielectric, uniform charge collection efficiency over large areas of pixels, and acceptable leakage current densities.Peer reviewe

Increased copy number at 3p14 in breast cancer

INTRODUCTION: The present study was conducted to investigate if chromosome band 3p14 is of any pathogenic significance in the malignant process of breast cancer. Genetic studies have implicated a tumour suppressor gene on chromosome arm 3p and we have proposed LRIG1 at 3p14 as a candidate tumour suppressor. The LRIG1 gene encodes an integral membrane protein that counteracts signalling by receptor tyrosine kinases belonging to the ERBB family. LRIG1 mRNA and protein are expressed in many tissues, including breast tissue. METHODS: In the present report we analysed the LRIG1 gene by fluorescence in situ hybridisation (FISH), LRIG1 mRNA by quantitative RT-PCR, and LRIG1 protein by western blot analysis. Two tumour series were analysed; one series consisted of 19 tumour samples collected between 1987 and 1995 and the other series consisted of 9 tumour samples with corresponding non-neoplastic breast tissues collected consecutively. RESULTS: The LRIG1 gene showed increased copy number in 11 out of 28 tumours (39%) and only one tumour showed a deletion at this locus. Increased LRIG1 copy number was associated with increased levels of LRIG1 mRNA (two of three tumours) and protein (four of four tumours) in the tumours compared to matched non-neoplastic breast tissue, as assessed by RT-PCR and western blot analysis. CONCLUSION: The molecular function of LRIG1 as a negative regulator of ERBB receptors questions the biological significance of increased LRIG1 copy number in breast cancer. We propose that a common, but hitherto unrecognised, breast cancer linked gene is located within an amplicon containing the LRIG1 locus at 3p14.3