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2017
Hussain, A, Semeano ATS, Palma SICJ, Pina AS, Almeida J, Medrado BF, Pádua ACCS, Carvalho AL, Dionísio M, Li RWC, Gamboa H, Ulijn RV, Gruber J, Roque ACA.  2017.  Tunable Gas Sensing Gels by Cooperative Assembly. Advanced Functional Materials. 27:1700803–n/a., Number 27 AbstractWebsite

The cooperative assembly of biopolymers and small molecules can yield functional materials with precisely tunable properties. Here, the fabrication, characterization, and use of multicomponent hybrid gels as selective gas sensors are reported. The gels are composed of liquid crystal droplets self-assembled in the presence of ionic liquids, which further coassemble with biopolymers to form stable matrices. Each individual component can be varied and acts cooperatively to tune gels' structure and function. The unique molecular environment in hybrid gels is explored for supramolecular recognition of volatile compounds. Gels with distinct compositions are used as optical and electrical gas sensors, yielding a combinatorial response conceptually mimicking olfactory biological systems, and tested to distinguish volatile organic compounds and to quantify ethanol in automotive fuel. The gel response is rapid, reversible, and reproducible. These robust, versatile, modular, pliant electro-optical soft materials possess new possibilities in sensing triggered by chemical and physical stimuli.

2016
Brás, JLA, Pinheiro BA, Cameron K, Cuskin F, Viegas A, Najmudin S, Bule P, Pires VMR, Romão MJ, Bayer EA, Spencer HL, Smith S, Gilbert HJ, Alves VD, Carvalho AL, Fontes CMGA.  2016.  Diverse specificity of cellulosome attachment to the bacterial cell surface, dec. Scientific Reports. 6:38292.: The Author(s) AbstractWebsite

During the course of evolution, the cellulosome, one of Nature's most intricate multi-enzyme complexes, has been continuously fine-tuned to efficiently deconstruct recalcitrant carbohydrates. To facilitate the uptake of released sugars, anaerobic bacteria use highly ordered protein-protein interactions to recruit these nanomachines to the cell surface. Dockerin modules located within a non-catalytic macromolecular scaffold, whose primary role is to assemble cellulosomal enzymatic subunits, bind cohesin modules of cell envelope proteins, thereby anchoring the cellulosome onto the bacterial cell. Here we have elucidated the unique molecular mechanisms used by anaerobic bacteria for cellulosome cellular attachment. The structure and biochemical analysis of five cohesin-dockerin complexes revealed that cell surface dockerins contain two cohesin-binding interfaces, which can present different or identical specificities. In contrast to the current static model, we propose that dockerins utilize multivalent modes of cohesin recognition to recruit cellulosomes to the cell surface, a mechanism that maximises substrate access while facilitating complex assembly.

Terao, M, Romão MJ, Leimkühler S, Bolis M, Fratelli M, Coelho C, Santos-Silva T, Garattini E.  2016.  Structure and function of mammalian aldehyde oxidases. Archives of Toxicology. 90:753–780., Number 4 AbstractWebsite

Mammalian aldehyde oxidases (AOXs; EC1.2.3.1) are a group of conserved proteins belonging to the family of molybdo-flavoenzymes along with the structurally related xanthine dehydrogenase enzyme. AOXs are characterized by broad substrate specificity, oxidizing not only aromatic and aliphatic aldehydes into the corresponding carboxylic acids, but also hydroxylating a series of heteroaromatic rings. The number of AOX isoenzymes expressed in different vertebrate species is variable. The two extremes are represented by humans, which express a single enzyme (AOX1) in many organs and mice or rats which are characterized by tissue-specific expression of four isoforms (AOX1, AOX2, AOX3, and AOX4). In vertebrates each AOX isoenzyme is the product of a distinct gene consisting of 35 highly conserved exons. The extant species-specific complement of AOX isoenzymes is the result of a complex evolutionary process consisting of a first phase characterized by a series of asynchronous gene duplications and a second phase where the pseudogenization and gene deletion events prevail. In the last few years remarkable advances in the elucidation of the structural characteristics and the catalytic mechanisms of mammalian AOXs have been made thanks to the successful crystallization of human AOX1 and mouse AOX3. Much less is known about the physiological function and physiological substrates of human AOX1 and other mammalian AOX isoenzymes, although the importance of these proteins in xenobiotic metabolism is fairly well established and their relevance in drug development is increasing. This review article provides an overview and a discussion of the current knowledge on mammalian AOX.

2015
De Schutter, A, Correia HD, Freire DM, Rivas MG, Rizzi A, Santos-Silva T, González PJ, Van Doorslaer S.  2015.  Ligand Binding to Chlorite Dismutase from Magnetospirillum sp, October. The journal of physical chemistry. B. 119:13859—13869., Number 43 AbstractWebsite
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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.

Correia, HD, Marangon J, Brondino CD, Moura JJG, Romao MJ, Gonzalez PJ, Santos-Silva T.  2015.  Aromatic aldehydes at the active site of aldehyde oxidoreductase from Desulfovibrio gigas: reactivity and molecular details of the enzyme-substrate and enzyme-product interaction. Journal of Biological Inorganic Chemistry. 20:219-229., Number 2 AbstractWebsite

Desulfovibrio gigas aldehyde oxidoreductase (DgAOR) is a mononuclear molybdenum-containing enzyme from the xanthine oxidase (XO) family, a group of enzymes capable of catalyzing the oxidative hydroxylation of aldehydes and heterocyclic compounds. The kinetic studies reported in this work showed that DgAOR catalyzes the oxidative hydroxylation of aromatic aldehydes, but not heterocyclic compounds. NMR spectroscopy studies using C-13-labeled benzaldehyde confirmed that DgAOR catalyzes the conversion of aldehydes to the respective carboxylic acids. Steady-state kinetics in solution showed that high concentrations of the aromatic aldehydes produce substrate inhibition and in the case of 3-phenyl propionaldehyde a suicide substrate behavior. Hydroxyl-substituted aromatic aldehydes present none of these behaviors but the kinetic parameters are largely affected by the position of the OH group. High-resolution crystallographic structures obtained from single crystals of active-DgAOR soaked with benzaldehyde showed that the side chains of Phe(425) and Tyr(535) are important for the stabilization of the substrate in the active site. On the other hand, the X-ray data of DgAOR soaked with trans-cinnamaldehyde showed a cinnamic acid molecule in the substrate channel. The X-ray data of DgAOR soaked with 3-phenyl propionaldehyde showed clearly how high substrate concentrations inactivate the enzyme by binding covalently at the surface of the enzyme and blocking the substrate channel. The different reactivity of DgAOR versus aldehyde oxidase and XO towards aromatic aldehydes and N-heterocyclic compounds is explained on the basis of the present kinetic and structural data.

Seixas, JD, Santos MFA, Mukhopadhyay A, Coelho AC, Reis PM, Veiros LF, Marques AR, Penacho N, Goncalves AML, Romao MJ, Bernardes GJL, Santos-Silva T, Romao CC.  2015.  A contribution to the rational design of Ru(CO)(3)Cl2L complexes for in vivo delivery of CO. Dalton Transactions. 44:5058-5075., Number 11 AbstractWebsite

A few ruthenium based metal carbonyl complexes, e.g. CORM-2 and CORM-3, have therapeutic activity attributed to their ability to deliver CO to biological targets. In this work, a series of related complexes with the formula [Ru(CO)(3)Cl2L] (L = DMSO (3), L-H3CSO(CH2)(2)CH(NH2)CO2H) (6a); D,L-H3CSO(CH2)(2)CH-(NH2)CO2H (6b); 3-NC5H4(CH2)(2)SO3.Na (7); 4-NC5H4(CH2)(2)SO3Na (8); PTA (9); DAPTA (10); H3CS-(CH2)(2)CH(OH) CO2H (11); CNCMe2CO2Me (12); CNCMeEtCO2Me (13); CN(c-C3H4)CO2Et) (14)) were designed, synthesized and studied. The effects of L on their stability, CO release profile, cytotoxicity and anti-inflammatory properties are described. The stability in aqueous solution depends on the nature of L as shown using HPLC and LC-MS studies. The isocyanide derivatives are the least stable complexes, and the S-bound methionine oxide derivative is the more stable one. The complexes do not release CO gas to the headspace, but release CO2 instead. X-ray diffraction of crystals of the model protein Hen Egg White Lysozyme soaked with 6b (4UWN) and 8 (4UWV) shows the addition of Ru-II(CO)(H2O)(4) at the His15 binding site. Soakings with 7 (4UWU) produced the metallacarboxylate [Ru(COOH)(CO)(H2O)(3)](+) bound to the His15 site. The aqueous chemistry of these complexes is governed by the water-gas shift reaction initiated with the nucleophilic attack of HO- on coordinated CO. DFT calculations show this addition to be essentially barrierless. The complexes have low cytotoxicity and low hemolytic indices. Following i.v. administration of CORM-3, the in vivo bio-distribution of CO differs from that obtained with CO inhalation or with heme oxygenase stimulation. A mechanism for CO transport and delivery from these complexes is proposed.

Cerqueira, NMFSA, Coelho C, Bras NF, Fernandes PA, Garattini E, Terao M, Romao MJ, Ramos MJ.  2015.  Insights into the structural determinants of substrate specificity and activity in mouse aldehyde oxidases. Journal of Biological Inorganic Chemistry. 20:209-217., Number 2 AbstractWebsite

In this work, a combination of homology modeling and molecular dynamics (MD) simulations was used to investigate the factors that modulate substrate specificity and activity of the mouse AOX isoforms: mAOX1, mAOX2 (previously mAOX3l1), mAOX3 and mAOX4. The results indicate that the AOX isoform structures are highly preserved and even more conserved than the corresponding amino acid sequences. The only differences are at the protein surface and substrate-binding site region. The substrate-binding site of all isoforms consists of two regions: the active site, which is highly conserved among all isoforms, and a isoform-specific region located above. We predict that mAOX1 accepts a broader range of substrates of different shape, size and nature relative to the other isoforms. In contrast, mAOX4 appears to accept a more restricted range of substrates. Its narrow and hydrophobic binding site indicates that it only accepts small hydrophobic substrates. Although mAOX2 and mAOX3 are very similar to each other, we propose the following pairs of overlapping substrate specificities: mAOX2/mAOX4 and mAOX3/mAXO1. Based on these considerations, we propose that the catalytic activity between all isoforms should be similar but the differences observed in the binding site might influence the substrate specificity of each enzyme. These results also suggest that the presence of several AOX isoforms in mouse allows them to oxidize more efficiently a wider range of substrates. This contrasts with the same or other organisms that only express one isoform and are less efficient or incapable of oxidizing the same type of substrates.

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.

2014
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.

Santos, MFA, Correia I, Oliveira AR, Garribba E, Pessoa JC, Santos-Silva T.  2014.  Vanadium Complexes as Prospective Therapeutics: Structural Characterization of a VIV Lysozyme Adduct. European Journal of Inorganic Chemistry. :n/a–n/a.: WILEY-VCH Verlag AbstractWebsite

The biological activity of vanadium complexes, namely, as insulin enhancers, is well known. We report a combined X-ray crystallography, electron paramagnetic resonance, and density functional theory study of the interaction of vanadium picolinate complexes with hen egg white lysozyme (HEWL). We show that the VIVO(pic)2 complex covalently binds to the COO– group of the side chain of Asp52 of HEWL. The long VIV=O bond obtained in the X-ray study is explained to be due to reduction of VIV to VIII during exposure of the crystals to the intense X-ray beam.

2013
Seixas, JD, Mukhopadhyay A, Santos-Silva T, Otterbein LE, Gallo DJ, Rodrigues SS, Guerreiro BH, Goncalves AML, Penacho N, Marques AR, Coelho AC, Reis PM, Romao MJ, Romao CC.  2013.  Characterization of a versatile organometallic pro-drug (CORM) for experimental CO based therapeutics. Dalton Transactions. 42:5985-5998., Number 17 AbstractWebsite
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Mehtab, S, Goncalves G, Roy S, Tomaz AI, Santos-Silva T, Santos MFA, Romao MJ, Jakusch T, Kiss T, Pessoa JC.  2013.  Interaction of vanadium(IV) with human serum apo-transferrin. Journal of Inorganic Biochemistry. 121:187-195. AbstractWebsite
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Marangon, J, Correia HD, Brondino CD, Moura JJG, Romao MJ, Gonzalez PJ, Santos-Silva T.  2013.  Kinetic and Structural Studies of Aldehyde Oxidoreductase from Desulfovibrio gigas Reveal a Dithiolene-Based Chemistry for Enzyme Activation and Inhibition by H2O2. Plos One. 8, Number 12 AbstractWebsite
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Verma, AK, Goyal A, Freire F, Bule P, Venditto I, Bras JLA, Santos H, Cardoso V, Bonifacio C, Thompson A, Romao MJ, Prates JAM, Ferreira LMA, Fontes CMGA, Najmudin S.  2013.  Overexpression, crystallization and preliminary X-ray crystallographic analysis of glucuronoxylan xylanohydrolase (Xyn30A) from Clostridium thermocellum. Acta Crystallographica Section F-Structural Biology and Crystallization Communications. 69:1440-1442. AbstractWebsite
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2012
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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Bras, JLA, Carvalho AL, Viegas A, Najmudin S, Alves VD, Prates JAM, Ferreira LMA, Romao MJ, Gilbert HJ, Fontes CMGA.  2012.  ESCHERICHIA COLI EXPRESSION, PURIFICATION, CRYSTALLIZATION, AND STRUCTURE DETERMINATION OF BACTERIAL COHESIN-DOCKERIN COMPLEXES. Cellulases. 510(Gilbert, H. J., Ed.).:395-415. Abstract
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Coelho, C, Mahro M, Trincao J, Carvalho ATP, Ramos MJ, Terao M, Garattini E, Leimkuehler S, Romao MJ.  2012.  The First Mammalian Aldehyde Oxidase Crystal Structure INSIGHTS INTO SUBSTRATE SPECIFICITY. Journal of Biological Chemistry. 287:40690-40702., Number 48 AbstractWebsite
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Bras, JLA, Alves VD, Carvalho AL, Najmudin S, Prates JAM, Ferreira LMA, Bolam DN, Romao MJ, Gilbert HJ, Fontes CMGA.  2012.  Novel Clostridium thermocellum Type I Cohesin-Dockerin Complexes Reveal a Single Binding Mode. Journal of Biological Chemistry. 287:44394-44405., Number 53 AbstractWebsite
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Aveiro, SS, Freire F, Clayton J, Cameloc M, Carvalho AL, Ferreira GC, Romao MJ, Macedo AL, Goodfellow BJ.  2012.  Structural studies of the p22HBP/SOUL family of heme-binding proteins. Febs Journal. 279:458-458. AbstractWebsite
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2011
Mahro, M, Coelho C, Trincao J, Rodrigues D, Terao M, Garattini E, Saggu M, Lendzian F, Hildebrandt P, Romao MJ, Leimkuehler S.  2011.  Characterization and Crystallization of Mouse Aldehyde Oxidase 3: From Mouse Liver to Escherichia coli Heterologous Protein Expression. Drug Metabolism and Disposition. 39:1939-1945., Number 10 AbstractWebsite
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Coelho, C, Gonzalez PJ, Moura JJG, Moura I, Trincao J, Romao MJ.  2011.  The Crystal Structure of Cupriavidus necator Nitrate Reductase in Oxidized and Partially Reduced States. Journal of Molecular Biology. 408:932-948., Number 5 AbstractWebsite
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Mukhopadhyay, A, Kladova AV, Bursakov SA, Gavel YO, Calvete JJ, Shnyrov VL, Moura I, Moura JJG, Romao MJ, Trincao J.  2011.  Crystal structure of the zinc-, cobalt-, and iron-containing adenylate kinase from Desulfovibrio gigas: a novel metal-containing adenylate kinase from Gram-negative bacteria. Journal of Biological Inorganic Chemistry. 16:51-61., Number 1 AbstractWebsite
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Garcia-Alvarez, B, Melero R, Dias FMV, Prates JAM, Fontes CMGA, Smith SP, Romao MJ, Carvalho AL, Llorca O.  2011.  Molecular Architecture and Structural Transitions of a Clostridium thermocellum Mini-Cellulosome. Journal of Molecular Biology. 407:571-580., Number 4 AbstractWebsite
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Bras, JLA, Cartmell A, Carvalho ALM, Verze G, Bayer EA, Vazana Y, Correia MAS, Prates JAM, Ratnaparkhe S, Boraston AB, Romao MJ, Fontes CMGA, Gilbert HJ.  2011.  Structural insights into a unique cellulase fold and mechanism of cellulose hydrolysis. Proceedings of the National Academy of Sciences of the United States of America. 108:5237-5242., Number 13 AbstractWebsite
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