Optimizing Electroactive Organisms: The Effect of Orthologous Proteins

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Superoxide reductase from Giardia intestinalis: structural characterization of the first sor from a eukaryotic organism shows an iron centre that is highly sensitive to photoreduction

Superoxide reductase (SOR), which is commonly found in prokaryotic organisms, affords protection from oxidative stress by reducing the superoxide anion to hydrogen peroxide. The reaction is catalyzed at the iron centre, which is highly conserved among the prokaryotic SORs structurally characterized to date. Reported here is the first structure of an SOR from a eukaryotic organism, the protozoan parasite Giardia intestinalis (GiSOR), which was solved at 2.0 Å resolution. By collecting several diffraction data sets at 100 K from the same flash-cooled protein crystal using synchrotron X-ray radiation, photoreduction of the iron centre was observed. Reduction was monitored using an online UV-visible microspectrophotometer, following the decay of the 647 nm absorption band characteristic of the iron site in the glutamate-bound, oxidized state. Similarly to other 1Fe-SORs structurally characterized to date, the enzyme displays a tetrameric quaternary-structure arrangement. As a distinctive feature, the N-terminal loop of the protein, containing the characteristic EKHxP motif, revealed an unusually high flexibility regardless of the iron redox state. At variance with previous evidence collected by X-ray crystallography and Fourier transform infrared spectroscopy of prokaryotic SORs, iron reduction did not lead to dissociation of glutamate from the catalytic metal or other structural changes; however, the glutamate ligand underwent X-ray-induced chemical changes, revealing high sensitivity of the GiSOR active site to X-ray radiation damage

Structure/function studies of the NAD⁺-dependent DNA ligase from the poly-extremophile Deinococcus radiodurans reveal importance of the BRCT domain for DNA binding

Bacterial NAD+-dependent DNA ligases (LigAs) are enzymes involved in replication, recombination, and DNA-repair processes by catalyzing the formation of phosphodiester bonds in the backbone of DNA. These multidomain proteins exhibit four modular domains, that are highly conserved across species, with the BRCT (breast cancer type 1 C-terminus) domain on the C-terminus of the enzyme. In this study, we expressed and purified both recombinant full-length and a C-terminally truncated LigA from Deinococcus radiodurans (DrLigA and DrLigA∆BRCT) and characterized them using biochemical and X-ray crystallography techniques. Using seeds of DrLigA spherulites, we obtained ≤ 100 µm plate crystals of DrLigA∆BRCT. The crystal structure of the truncated protein was obtained at 3.4 Å resolution, revealing DrLigA∆BRCT in a non-adenylated state. Using molecular beacon-based activity assays, we demonstrated that DNA ligation via nick sealing remains unaffected in the truncated DrLigA∆BRCT. However, DNA-binding assays revealed a reduction in the affinity of DrLigA∆BRCT for dsDNA. Thus, we conclude that the flexible BRCT domain, while not critical for DNA nick-joining, plays a role in the DNA binding process, which may be a conserved function of the BRCT domain in LigA-type DNA ligases

Structural Basis of RICs Iron Donation for Iron-Sulfur Cluster Biogenesis

Funding Information: We thank L?gia S. Nobre and Joana M. Baptista for contribution at the initial stage of the work, and Cl?udia S. Freitas for technical support. We also thank Professor Miguel Teixeira of ITQB-NOVA for critical reading of the manuscript. We thank the XALOC staff and floor coordinators at the synchrotron ALBA for the YtfEM data collection. We acknowledge the ESRF for provision of synchrotron radiation facilities and we would like to thank Gianluca Santoni for assistance using the beamline ID30A-3 for the YtfEM-E159L data collection. We also thank Diamond Light Source for beamtime and the staff of beamline I04 for assistance with crystal testing and data collection of YtfEM-E125L. Funding. This work was financially supported by Funda??o para a Ci?ncia e Tecnologia (Portugal) through fellowship SFRH/BD/118545/2016 (LOS) and R&D unit LISBOA-01-0145-FEDER007660 (MostMicro) co-funded by FCT/MCTES and FEDER funds under the PT2020 Partnership Agreement. This work was partially supported by PPBI ? Portuguese Platform of BioImaging (PPBI-POCI-01-0145-FEDER-022122) co-funded by national funds from OE ? ?Or?amento de Estado? and by European funds from FEDER ? ?Fundo Europeu de Desenvolvimento Regional.? We also acknowledge funding from the European Union?s Horizon 2020 Research and Innovation Program under grant agreement no. 810856. Publisher Copyright: © Copyright © 2021 Silva, Matias, Romão and Saraiva. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.Escherichia coli YtfE is a di-iron protein of the widespread Repair of Iron Centers proteins (RIC) family that has the capacity to donate iron, which is a crucial component of the biogenesis of the ubiquitous family of iron-sulfur proteins. In this work we identify in E. coli a previously unrecognized link between the YtfE protein and the major bacterial system for iron-sulfur cluster (ISC) assembly. We show that YtfE establishes protein-protein interactions with the scaffold IscU, where the transient cluster is formed, and the cysteine desulfurase IscS. Moreover, we found that promotion by YtfE of the formation of an Fe-S cluster in IscU requires two glutamates, E125 and E159 in YtfE. Both glutamates form part of the entrance of a protein channel in YtfE that links the di-iron center to the surface. In particular, E125 is crucial for the exit of iron, as a single mutation to leucine closes the channel rendering YtfE inactive for the build-up of Fe-S clusters. Hence, we provide evidence for the key role of RICs as bacterial iron donor proteins involved in the biogenesis of Fe-S clusters.publishersversionpublishe

Neotectónica e períodos de recorrência de grandes sismos e tsunamis na margem SW Ibérica e Golfo de Cádis

Neste trabalho usámos o método numérico das placas finas (Bird, 1999) para modelar a Neotectónica no Golfo de Cadiz e estimar os períodos de recorrência de grandes sismos e tsunamis. Foram testadas várias configurações de falhas e condições fronteira, e os resultados comparados com as observações de GPS, tensão e deformação sísmica. O melhor ajuste às observações é obtido com um modelo que apresenta uma taxa de movimentação de 1 mm/a nos cavalgamentos com orientação E-O e NE-SO, o que corresponde a períodos de recorrência de 1150, 3620 e 9900 anos para sismos de magnitude Mw de 7, 8 e 8.75

High-resolution structure of an atypical α-phosphoglucomutase related to eukaryotic phosphomannomutases

The first structure of a bacterial α-phosphoglucomutase with an overall fold similar to eukaryotic phosphomannomutases is reported. Unlike most α-phosphoglucomutases within the α-D-phosphohexomutase superfamily, it belongs to subclass IIb of the haloacid dehalogenase superfamily (HADSF). It catalyzes the reversible conversion of α-glucose 1-phosphate to glucose 6-phosphate. The crystal structure of α-phosphoglucomutase from Lactococcus lactis (APGM) was determined at 1.5 Å resolution and contains a sulfate and a glycerol bound at the enzyme active site that partially mimic the substrate. A dimeric form of APGM is present in the crystal and in solution, an arrangement that may be functionally relevant. The catalytic mechanism of APGM and its strict specificity towards α-glucose 1-phosphate are discussed.Diamond Light Source

Redox-Polymer-Wired [NiFeSe] Hydrogenase Variants with Enhanced O2 Stability for Triple-Protected High-Current-Density H2-Oxidation Bioanodes

Variants of the highly active [NiFeSe] hydrogenase from D. vulgaris Hildenborough that exhibit enhanced O2 tolerance were used as H2-oxidation catalysts in H2/O2 biofuel cells. Two [NiFeSe] variants were electrically wired by means of low-potential viologen-modified redox polymers and evaluated with respect to H2-oxidation and stability against O2 in the immobilized state. The two variants showed maximum current densities of (450±84) μA cm−2 for G491A and (476±172) μA cm−2 for variant G941S on glassy carbon electrodes and a higher O2 tolerance than the wild type. In addition, the polymer protected the enzyme from O2 damage and high-potential inactivation, establishing a triple protection for the bioanode. The use of gas-diffusion bioanodes provided current densities for H2-oxidation of up to 6.3 mA cm−2. Combination of the gas-diffusion bioanode with a bilirubin oxidase-based gas-diffusion O2-reducing biocathode in a membrane-free biofuel cell under anode-limiting conditions showed unprecedented benchmark power densities of 4.4 mW cm−2 at 0.7 V and an open-circuit voltage of 1.14 V even at moderate catalyst loadings, outperforming the previously reported system obtained with the [NiFeSe] wild type and the [NiFe] hydrogenase from D. vulgaris Miyazaki F.inpres