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Oliveira, AR, Mota C, Romão MJ, Pereira IAC.  2022.  The W/SeCys-FdhAB formate dehydrogenase from Desulfovibrio vulgaris Hildenborough, 2022/06/10. Encyclopedia of Inorganic and Bioinorganic Chemistry. :1-12. Abstract

Abstract The W/SeCys-FdhAB formate dehydrogenase from Desulfovibrio vulgaris Hildenborough is a dimeric periplasmic enzyme that catalyzes the reversible oxidation of formate and reduction of CO2. It belongs to the group of metal-dependent FDHs, with a tungsten at the active site coordinated by two pyranopterin guanine dinucleotides, a selenocysteine, and one labile sulfur atom. FdhAB has a remarkably high activity and unusual tolerance to oxygen, making it an ideal model system to study biological CO2 reduction.

Otrelo-Cardoso, AR, da Silva Correia MA, Schwuchow V, Svergun DI, Romao MJ, Leimkuehler S, Santos-Silva T.  2014.  Structural Data on the Periplasmic Aldehyde Oxidoreductase PaoABC from Escherichia coli: SAXS and Preliminary X-ray Crystallography Analysis. International Journal of Molecular Sciences. 15:2223-2236., Number 2 AbstractWebsite
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Otrelo-Cardoso, AR, Schwuchow V, Rodrigues D, Cabrita EJ, Leimkuehler S, Romao MJ, Santos-Silva T.  2014.  Biochemical, Stabilization and Crystallization Studies on a Molecular Chaperone (PaoD) Involved in the Maturation of Molybdoenzymes. Plos One. 9, Number 1 AbstractWebsite
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Otrelo-Cardoso, AR, Nair RR, Correia MAS, Cordeiro RCS, Panjkovich A, Svergun DI, Santos-Silva T, Rivas MG.  2017.  Highly selective tungstate transporter protein TupA from Desulfovibrio alaskensis G20, 2017. Scientific Reports. 7(1):5798. AbstractWebsite

Molybdenum and tungsten are taken up by bacteria and archaea as their soluble oxyanions through high affinity transport systems belonging to the ATP-binding cassette (ABC) transporters. The component A (ModA/TupA) of these transporters is the first selection gate from which the cell differentiates between MoO4 2−, WO4 2− and other similar oxyanions. We report the biochemical characterization and the crystal structure of the apo-TupA from Desulfovibrio desulfuricans G20, at 1.4 Å resolution. Small Angle X-ray Scattering data suggests that the protein adopts a closed and more stable conformation upon ion binding. The role of the arginine 118 in the selectivity of the oxyanion was also investigated and three mutants were constructed: R118K, R118E and R118Q. Isothermal titration calorimetry clearly shows the relevance of this residue for metal discrimination and oxyanion binding. In this sense, the three variants lost the ability to coordinate molybdate and the R118K mutant keeps an extremely high affinity for tungstate. These results contribute to an understanding of the metal-protein interaction, making it a suitable candidate for a recognition element of a biosensor for tungsten detection.

Otrelo-Cardoso, AR, Nair RR, Correia MAS, Rivas MG, Santos-Silva T.  2014.  TupA: A Tungstate Binding Protein in the Periplasm of Desulfovibrio alaskensis G20, 2014/05/29/accep. International Journal of Molecular Sciences. 15(7):11783-11798.: MDPI AbstractWebsite

The TupABC system is involved in the cellular uptake of tungsten and belongs to the ABC (ATP binding cassette)-type transporter systems. The TupA component is a periplasmic protein that binds tungstate anions, which are then transported through the membrane by the TupB component using ATP hydrolysis as the energy source (the reaction catalyzed by the ModC component). We report the heterologous expression, purification, determination of affinity binding constants and crystallization of the Desulfovibrio alaskensis G20 TupA. The tupA gene (locus tag Dde_0234) was cloned in the pET46 Enterokinase/Ligation-Independent Cloning (LIC) expression vector, and the construct was used to transform BL21 (DE3) cells. TupA expression and purification were optimized to a final yield of 10 mg of soluble pure protein per liter of culture medium. Native polyacrylamide gel electrophoresis was carried out showing that TupA binds both tungstate and molybdate ions and has no significant interaction with sulfate, phosphate or perchlorate. Quantitative analysis of metal binding by isothermal titration calorimetry was in agreement with these results, but in addition, shows that TupA has higher affinity to tungstate than molybdate. The protein crystallizes in the presence of 30% (w/v) polyethylene glycol 3350 using the hanging-drop vapor diffusion method. The crystals diffract X-rays beyond 1.4 Å resolution and belong to the P2(1) space group, with cell parameters a = 52.25 Å, b = 42.50 Å, c = 54.71 Å, β = 95.43°. A molecular replacement solution was found, and the structure is currently under refinement.

Outis, M, Rosa V, Laia CAT, Lima JC, Barroso S, Carvalho AL, Calhorda MJ, Avilés T.  2020.  Synthesis, Crystal Structure, and DFT Study of Two New Dinuclear Copper(I) Complexes Bearing Ar-BIAN Ligands Functionalized with NO2 Groups. European Journal of Inorganic Chemistry. 2020:2900-2911., Number 30 AbstractWebsite

{Two new bis(aryl-imino)-acenaphthene, Ar-BIAN (Ar = 2

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Palma, AS, Liu Y, Zhang Y, Zhang H, Luis AS, Carvalho AL, Gilbert HJ, Boraston A, Fontes CMGA, Chai W, Ten F.  2012.  Designer-oligosaccharide microarrays to decipher ligands in mammalian and prokaryotic glucan-recognition systems. Glycobiology. 22:1612-1613., Number 11 AbstractWebsite
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Palma, AS, Pinheiro B, Liu Y, Takeda Y, Chai W, Ito Y, Romao MJ, Carvalho AL, Feizi T.  2013.  The Structural Basis of the Recognition of Di-glucosylated N-glycans by the ER Lectin Malectin. Glycobiology. 23:1368-1369., Number 11 AbstractWebsite
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Palma, AS, Liu Y, Zhang H, Zhang Y, McCleary BV, Yu G, Huang Q, Guidolin LS, Ciocchini AE, Torosantucci A, Wang D, Carvalho AL, Fontes CM, Mulloy B, Childs RA, Feizi T, Chai W.  2015.  Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry. Mol Cell Proteomics. AbstractWebsite

Glucans are polymers of D-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes including immunomodulation, anti-cancer activities, pathogen virulence and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure-function studies and their exploitation. We describe construction of a glucome microarray, the first sequence-defined glycome-scale microarray, using a designer approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. The negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear homo and hetero and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signalling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.

Peixoto, D, Malta G, Cruz H, Barroso S, Carvalho AL, Ferreira LM, Branco PS.  2019.  N-Heterocyclic olefin catalysis for the ring opening of cyclic amidine compounds: a pathway to the synthesis of ε-caprolactam and γ-lactam-derived amines, 2019. The Journal of Organic Chemistry. : American Chemical Society AbstractWebsite

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Pessoa, JC, Gonçalves G, Roy S, Correia I, Mehtab S, Santos MFA, Santos-Silva T.  2014.  New insights on vanadium binding to human serum transferrin. Inorganica Chimica Acta. 420:60-68. AbstractWebsite

Abstract The knowledge on the binding of vanadium ions and complexes to serum proteins and how vanadium might be transported in blood and up-taken by cells has received much attention during the last decade, particularly as far as the transport of VIVO2+ is concerned. In this work we revise and discuss some relevant aspects of previous research, namely the two main types of binding proposed for transport of VIVO(carrier)2 complexes. New results, obtained by circular dichroism (CD), \{EPR\} and gel electrophoresis, regarding the binding of vanadium to hTF in the oxidation states +5 and +3 are also presented. Namely, evidences for the binding of VV-species to diferric-transferrin, designated by (FeIII)2hTF, as well as to (AlIII)2hTF, are presented and discussed, the possibility of up-take of vanadate by cells through (FeIII)2hTF endocytosis being suggested. It is also confirmed that \{VIII\} binds strongly to hTF, forming di-vanadium(III)-transferrin, designated by (VIII)2hTF, and gel electrophoresis experiments indicate that (VIII)2hTF corresponds to a ‘closed conformation’ similar to (FeIII)2hTF.

Pessoa, JC, Garribba E, Santos MFA, Santos-Silva T.  2015.  Vanadium and proteins: Uptake, transport, structure, activity and function, 2015/10/15/. The Ninth International Symposium on the Chemistry and Biological Chemistry of Vanadium. 301–302:49-86. AbstractWebsite

AbstractVanadium is an element ubiquitously present in our planet's crust and thus there are several organisms that use vanadium for activity or function of proteins. Examples are the vanadium-dependent haloperoxidases and the vanadium-containing nitrogenases. Some organisms that use vanadium have extremely efficient and selective protein-dependent systems for uptake and transport of vanadium and are able to accumulate high levels of vanadium from seawater, vanabins being a unique family of vanadium binding proteins found in ascidians involved in this process. For all of the systems a discussion regarding the role of the V-containing proteins is provided, mostly centered on structural aspects of the vanadium site and, when possible or relevant, relating this to the mechanisms operating. Phosphate is very important in biological systems and is involved in an extensive number of biological recognition and bio-catalytic systems. Vanadate(V) is able to inhibit many of the enzymes involved in these processes, such as ATPases, phosphatases, ribonucleases, phosphodiesterases, phosphoglucomutase and glucose-6-phosphatase, and it appears clear that this is closely related to the analogous physicochemical properties of vanadate and phosphate. The ability of vanadium to interfere with the metabolic processes involving Ca2+ and Mg2+, connected with its versatility to undergo changes in coordination geometry, allow V to influence the function of a large variety of phosphate-metabolizing enzymes and vanadate(V) salts and compounds have been frequently used either as inhibitors of these enzymes, or as probes to study the mechanisms of their reactions and catalytic cycle. In this review we give an overview of the many examples so far reported, also disclosing that vanadate(IV) may also have an equally efficient inhibiting effect. The prospective application of vanadium compounds as therapeutics has also been an important topic of research. How vanadium may be transported in blood and up-taken by cells are particularly relevant issues, this being mainly dependent on transferrin (and albumin) present in blood plasma. The thousands of studies reported on the effects of vanadium compounds reflect the complexity of the interactions occurring. Although it is not easy to anticipate/determine if a particular effect observed in a test tube or in vitro is also going to take place in vivo, it is clear that vanadium ions may interfere with many metabolic processes at many distinct levels. Emphasis is given on structural and functional aspects of vanadium–protein interactions relevant for vanadium binding and/or for clarification of role of the metal center in the reaction mechanisms. The additional knowledge that the presence of vanadium can change the action of a protein, other than simply inhibiting it, may also be important to understand how vanadium affects biological systems. This possibility, together with the vanadate–phosphate analogy further potentiates the belief that vanadium probably has relevant functions in living beings, which may involve interaction or incorporation of the metal ion and/or its compounds with several proteins.

Pinheiro, BA, Carvalho AL, Romao MJ, Fontes CM.  2013.  Study of the cohesin-dockerin interaction and its role in the C. thermocellum cellulosome assembly. European Biophysics Journal with Biophysics Letters. 42:S180-S180. AbstractWebsite
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Pires, VMR, Pereira PMM, Brás JLA, Correia M, Cardoso V, Bule P, Alves VD, Najmudin S, Venditto I, Ferreira LMA, Romão MJ, Carvalho AL, Fontes CMGA, Prazeres DM.  2017.  Stability and ligand promiscuity of type A carbohydrate-binding modules are illustrated by the structure of Spirochaeta thermophila StCBM64C, mar. Journal of Biological Chemistry. 292:4847–4860., Number 12 AbstractWebsite

Deconstruction of cellulose, the most abundant plant cell wall polysaccharide, requires the cooperative activity of a large repertoire of microbial enzymes. Modular cellulases contain non-catalytic type A Carbohydrate-Binding Modules (CBMs) that specifically bind to the crystalline regions of cellulose, thus promoting enzyme efficacy through proximity and targeting effects. Although type A CBMs play a critical role in cellulose recycling, their mechanism of action remains poorly understood. Here we produced a library of recombinant CBMs representative of the known diversity of type A modules. The binding properties of 40 CBMs, in fusion with an N-terminal green fluorescence protein (GFP) domain, revealed that type A CBMs possess the ability to recognize different crystalline forms of cellulose and chitin over a wide range of temperatures, pHs and ionic strengths. A Spirochaeta thermophila CBM64, in particular, displayed plasticity in its capacity to bind both crystalline and soluble carbohydrates under a wide range of extreme conditions. The structure of S. thermophila StCBM64C revealed an untwisted, flat, carbohydrate-binding interface comprising the side chains of four tryptophan residues in a coplanar linear arrangement. Significantly, two highly conserved asparagine side chains, each one located between two tryptophan residues, are critical to insoluble and soluble glucan recognition but not to bind xyloglucan. Thus, CBM64 compact structure and its extended and versatile ligand interacting platform illustrates how type A CBMs target their appended plant cell wall degrading enzymes to a diversity of recalcitrant carbohydrates under a wide range of environmental conditions.

Polino, M, Rho HS, Pina MP, Mallada R, Carvalho AL, Romão MJ, Coelhoso I, Gardeniers JGE, Crespo JG, Portugal CAM.  2021.  Protein Crystallization in a Microfluidic Contactor with Nafion®117 Membranes. Membranes. 11, Number 8 AbstractWebsite

Protein crystallization still remains mostly an empirical science, as the production of crystals with the required quality for X-ray analysis is dependent on the intensive screening of the best protein crystallization and crystal’s derivatization conditions. Herein, this demanding step was addressed by the development of a high-throughput and low-budget microfluidic platform consisting of an ion exchange membrane (117 Nafion® membrane) sandwiched between a channel layer (stripping phase compartment) and a wells layer (feed phase compartment) forming 75 independent micro-contactors. This microfluidic device allows for a simultaneous and independent screening of multiple protein crystallization and crystal derivatization conditions, using Hen Egg White Lysozyme (HEWL) as the model protein and Hg2+ as the derivatizing agent. This microdevice offers well-regulated crystallization and subsequent crystal derivatization processes based on the controlled transport of water and ions provided by the 117 Nafion® membrane. Diffusion coefficients of water and the derivatizing agent (Hg2+) were evaluated, showing the positive influence of the protein drop volume on the number of crystals and crystal size. This microfluidic system allowed for crystals with good structural stability and high X-ray diffraction quality and, thus, it is regarded as an efficient tool that may contribute to the enhancement of the proteins’ crystals structural resolution.

Polino, M, Carvalho AL, Juknaitė L, Portugal CAM, Coelhoso IM, Romão MJ, Crespo JG.  2017.  Ion-Exchange Membranes for Stable Derivatization of Protein Crystals, 2017. Crystal Growth & DesignCrystal Growth & Design. : American Chemical Society AbstractWebsite
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Raaijmakers, HCA, Romao MJ.  2006.  Formate-reduced E-coli formate dehydrogenase H: the reinterpretation of the crystal structure suggests a new reaction mechanism. Journal of Biological Inorganic Chemistry. 11:849-854., Number 7 AbstractWebsite
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Raaijmakers, H, Macieira S, Dias JM, Teixeira S, Bursakov S, Huber R, Moura JJG, Moura I, Romao MJ.  2002.  Gene sequence and the 1.8 angstrom crystal structure of the tungsten-containing formate dehydrogenase from Desulfolvibrio gigas. Structure. 10:1261-1272., Number 9 AbstractWebsite
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Raaijmakers, H, Teixeira S, Dias JM, Almendra MJ, Brondino CD, Moura I, Moura JJG, Romao MJ.  2001.  Tungsten-containing formats dehydrogenase from Desulfovibrio gigas: metal identification and preliminary structural data by multi-wavelength crystallography. Journal of Biological Inorganic Chemistry. 6:398-404., Number 4 AbstractWebsite
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Rebelo, JM, Dias JM, Huber R, Moura JJG, Romao MJ.  2001.  Structure refinement of the aldehyde oxidoreductase from Desulfovibrio gigas (MOP) at 1.28 angstrom. Journal of Biological Inorganic Chemistry. 6:791-800., Number 8 AbstractWebsite
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Rebelo, J, Macieira S, Dias JM, Huber R, Ascenso CS, Rusnak F, Moura JJG, Moura I, Romao MJ.  2000.  Gene sequence and crystal structure of the aldehyde oxidoreductase from Desulfovibrio desulfuricans ATCC 27774. Journal of Molecular Biology. 297:135-146., Number 1 AbstractWebsite
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Ribeiro, DO, Viegas A, Pires VMR, Medeiros-Silva J, Bule P, Chai W, Marcelo F, Fontes CMGA, Cabrita EJ, Palma AS, Carvalho AL.  2020.  Molecular basis for the preferential recognition of β1,3-1,4-glucans by the family 11 carbohydrate-binding module from Clostridium thermocellum. The FEBS Journal. 287:2723-2743., Number 13 AbstractWebsite

Understanding the specific molecular interactions between proteins and β1,3-1,4-mixed-linked d-glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognize them. The family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11) is known for its binding preference for β1,3-1,4-mixed-linked over β1,4-linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic biomass, the molecular determinants of its ligand specificity are not well defined. In this report, a combined approach of methodologies was used to unravel, at a molecular level, the ligand recognition of CtCBM11. The analysis of the interaction by carbohydrate microarrays and NMR and the crystal structures of CtCBM11 bound to β1,3-1,4-linked glucose oligosaccharides showed that both the chain length and the position of the β1,3-linkage are important for recognition, and identified the tetrasaccharide Glcβ1,4Glcβ1,4Glcβ1,3Glc sequence as a minimum epitope required for binding. The structural data, along with site-directed mutagenesis and ITC studies, demonstrated the specificity of CtCBM11 for the twisted conformation of β1,3-1,4-mixed-linked glucans. This is mediated by a conformation–selection mechanism of the ligand in the binding cleft through CH-π stacking and a hydrogen bonding network, which is dependent not only on ligand chain length, but also on the presence of a β1,3-linkage at the reducing end and at specific positions along the β1,4-linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed-linked β-glucans strengthens its exploitation for the design of new biomolecules with improved capabilities and applications in health and agriculture. Database Structural data are available in the Protein Data Bank under the accession codes 6R3M and 6R31.

Ribeiro, T, Santos-Silva T, Alves VD, Dias FMV, Luis AS, Prates JAM, Ferreira LMA, Romao MJ, Fontes CMGA.  2010.  Family 42 carbohydrate-binding modules display multiple arabinoxylan-binding interfaces presenting different ligand affinities. Biochimica Et Biophysica Acta-Proteins and Proteomics. 1804:2054-2062., Number 10 AbstractWebsite
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Ribeiro, DO, Pinheiro BA, Carvalho AL, Palma AS.  2018.  Targeting protein-carbohydrate interactions in plant cell-wall biodegradation: the power of carbohydrate microarrays. Carbohydrate Chemistry: Chemical and Biological Approaches Volume 43. 43:159-176.: The Royal Society of Chemistry Abstract

The plant cell-wall is constituted by structurally diverse polysaccharides. The biodegradation of these is a crucial process for life sustainability. Cellulolytic microorganisms are highly efficient in this process by assembling modular architectures of carbohydrate-active enzymes with appended non-catalytic carbohydrate-binding modules (CBMs). Carbohydrate microarrays offer high-throughput and sensitive tools for uncovering carbohydrate-binding specificities of CBMs{,} which is pivotal to understand the function of these modules in polysaccharide biodegradation mechanisms. Features of this technology will be here briefly reviewed with highlights of microarray approaches to study plant-carbohydrates and CBM-carbohydrate interactions{,} along with an overview of plant polysaccharides and microorganisms strategies for their recognition.

Ribeiro, D, Kulakova A, Quaresma P, Pereira E, Bonifacio C, Romao MJ, Franco R, Carvalho AL.  2014.  Use of Gold Nanoparticles as Additives in Protein Crystallization. Crystal Growth & Design. 14:222-227., Number 1 AbstractWebsite

Gold nanoparticles (AuNPs) exhibit unique properties that have made them a very attractive material for application in biological assays. Given the potentially interesting interactions between AuNPs and biological macromolecules, we investigated AuNPs-induced protein crystal growth. Differently functionalized AuNPs were tested as additives in cocrystallization studies with model proteins (hen egg white lysozyme (HEWL), ribonuclease A (RNase A), and proteinase K) as well as with case studies where there were problems in obtaining well-diffracting crystals. Trials were performed considering different crystallization drawbacks, from total absence of crystals to improvement of crystal morphology, size, twinning, and number of crystals per drop. Improvement of some of these factors was observed in the cases of HEWL, RNase A, phenylalanine hydroxylase (PAR), myoglobin, native aldehyde oxidase (AOH), and human albumin. In these proteins, the presence of the AuNPs promoted an increase in the size and/or better crystal morphology. From the systematic trials and subsequent observations, it can be concluded that the introduction of AuNPs should definitely be considered in crystal optimization trials to improve previously determined crystallization conditions.