Publications

Export 207 results:
Sort by: Author Title [ Type  (Asc)] Year
Book Chapter
Mota, C, Santos Silva T, Terao M, Garattini E, Romão MJ, Leimkuehler S.  2019.  Aldehyde Oxidases as Enzymes in Phase I Drug Metabolism. Pharmaceutical Biocatalysis. (Peter Grunwald, Ed.)., New York: Jenny Stanford Publishing
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
n/a
Correia, VG, Pinheiro BA, Carvalho AL, Palma AS.  2019.  Resistance to Aminoglycosides. Antibiotic Drug Resistance. :1-38.: John Wiley & Sons, Ltd Abstract

Summary The emergence of bacterial resistance to different antibiotics in clinical use, together with the knowledge on the mechanisms by which bacteria resist the action of aminoglycosides, have contributed to the renewed interest in these molecules as potential antimicrobials. Here, we give an overview on natural and semisynthetic aminoglycosides and their structural features and modes of action, focusing on the structural insight underlying resistance mechanisms. Developments on carbohydrate chemistry and microarray technology are highlighted as powerful approaches toward generation of new aminoglycosides and for screening their interactions with RNAs and proteins. The link between antibiotic uptake and the human gut microbiome is also addressed, focusing on gut microbiome function and composition, antibiotic-induced alterations in host health, and antibiotic resistance. In addition, strategies to modulate human microbiome responses to antibiotics are discussed as novel approaches for aminoglycoside usage and for the effectiveness of antibiotic therapy.

Romao, MJ.  2006.  The role of molybdenum in biology. Metal Ions in Biology and Medicine, Vol 9. 9(Alpoim, M.C., Morais, P.V., Eds.).:507-510. Abstract
n/a
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.

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.

Journal Article
Najmudin, S, Pauleta SR, Moura I, Romao MJ.  2010.  The 1.4 angstrom resolution structure of Paracoccus pantotrophus pseudoazurin. Acta Crystallographica Section F-Structural Biology and Crystallization Communications. 66:627-635. AbstractWebsite
n/a
Varela, PF, Romero A, Sanz L, Romao MJ, Topfer-Petersen E, Calvete JJ.  1997.  The 2.4 angstrom resolution crystal structure of boar seminal plasma PSP-I/PSP-II: a zona pellucida-binding glycoprotein heterodimer of the spermadhesin family built by a CUB domain architecture. Journal of Molecular Biology. 274:635-649., Number 4 AbstractWebsite
n/a
Gonçalves, AM, Sousa Â, Pedro AQ, Romão MJ, Queiroz JA, Gallardo E, Passarinha LA.  2022.  Advances in Membrane-Bound Catechol-O-Methyltransferase Stability Achieved Using a New Ionic Liquid-Based Storage Formulation. International Journal of Molecular Sciences. 23, Number 13 AbstractWebsite

Membrane-bound catechol-O-methyltransferase (MBCOMT), present in the brain and involved in the main pathway of the catechol neurotransmitter deactivation, is linked to several types of human dementia, which are relevant pharmacological targets for new potent and nontoxic inhibitors that have been developed, particularly for Parkinson’s disease treatment. However, the inexistence of an MBCOMT 3D-structure presents a blockage in new drugs’ design and clinical studies due to its instability. The enzyme has a clear tendency to lose its biological activity in a short period of time. To avoid the enzyme sequestering into a non-native state during the downstream processing, a multi-component buffer plays a major role, with the addition of additives such as cysteine, glycerol, and trehalose showing promising results towards minimizing hMBCOMT damage and enhancing its stability. In addition, ionic liquids, due to their virtually unlimited choices for cation/anion paring, are potential protein stabilizers for the process and storage buffers. Screening experiments were designed to evaluate the effect of distinct cation/anion ILs interaction in hMBCOMT enzymatic activity. The ionic liquids: choline glutamate [Ch][Glu], choline dihydrogen phosphate ([Ch][DHP]), choline chloride ([Ch]Cl), 1- dodecyl-3-methylimidazolium chloride ([C12mim]Cl), and 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) were supplemented to hMBCOMT lysates in a concentration from 5 to 500 mM. A major potential stabilizing effect was obtained using [Ch][DHP] (10 and 50 mM). From the DoE 146% of hMBCOMT activity recovery was obtained with [Ch][DHP] optimal conditions (7.5 mM) at −80 °C during 32.4 h. These results are of crucial importance for further drug development once the enzyme can be stabilized for longer periods of time.

Oliveira, AR, Mota C, Vilela-Alves G, Manuel RR, Pedrosa N, Fourmond V, Klymanska K, Léger C, Guigliarelli B, Romão MJ, Cardoso Pereira IA.  2024.  An allosteric redox switch involved in oxygen protection in a CO2 reductase, 2024. 20(1):111-119. AbstractWebsite

Metal-dependent formate dehydrogenases reduce CO2 with high efficiency and selectivity, but are usually very oxygen sensitive. An exception is Desulfovibrio vulgaris W/Sec-FdhAB, which can be handled aerobically, but the basis for this oxygen tolerance was unknown. Here we show that FdhAB activity is controlled by a redox switch based on an allosteric disulfide bond. When this bond is closed, the enzyme is in an oxygen-tolerant resting state presenting almost no catalytic activity and very low formate affinity. Opening this bond triggers large conformational changes that propagate to the active site, resulting in high activity and high formate affinity, but also higher oxygen sensitivity. We present the structure of activated FdhAB and show that activity loss is associated with partial loss of the metal sulfido ligand. The redox switch mechanism is reversible in vivo and prevents enzyme reduction by physiological formate levels, conferring a fitness advantage during O2 exposure.

Glatigny, A, Hof P, Romao MJ, Huber R, Scazzocchio C.  1998.  Altered specificity mutations define residues essential for substrate positioning in xanthine dehydrogenase. Journal of Molecular Biology. 278:431-438., Number 2 AbstractWebsite
n/a
Moura, JJG, Goodfellow BJ, Romao MJ, Rusnak F, Moura I.  1996.  Analysis, design and engineering of simple iron-sulfur proteins: Tales from rubredoxin and desulforedoxin. Comments on Inorganic Chemistry. 19:47-+., Number 1 AbstractWebsite
n/a
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.

Engrola, F, Correia MAS, Watson C, Romão CC, Veiros LF, Romão MJ, Santos-Silva T, Santini JM.  2023.  Arsenite oxidase in complex with antimonite and arsenite oxyanions: Insights into the catalytic mechanism, 2023. Journal of Biological ChemistryJournal of Biological Chemistry. 299(8): Elsevier AbstractWebsite

Arsenic contamination of groundwater is among one of the biggest health threats affecting millions of people in the world. There is an urgent need for efficient arsenic biosensors where the use of arsenic metabolizing enzymes can be explored. In this work, we have solved four crystal structures of arsenite oxidase (Aio) in complex with arsenic and antimony oxyanions and the structures determined correspond to intermediate states of the enzymatic mechanism. These structural data were complemented with density-functional theory calculations providing a unique view of the molybdenum active site at different time points that, together with mutagenesis data, enabled to clarify the enzymatic mechanism and the molecular determinants for the oxidation of As(III) to the less toxic As(V) species.Arsenic contamination of groundwater is among one of the biggest health threats affecting millions of people in the world. There is an urgent need for efficient arsenic biosensors where the use of arsenic metabolizing enzymes can be explored. In this work, we have solved four crystal structures of arsenite oxidase (Aio) in complex with arsenic and antimony oxyanions and the structures determined correspond to intermediate states of the enzymatic mechanism. These structural data were complemented with density-functional theory calculations providing a unique view of the molybdenum active site at different time points that, together with mutagenesis data, enabled to clarify the enzymatic mechanism and the molecular determinants for the oxidation of As(III) to the less toxic As(V) species.

Bule, P, Alves VD, Israeli-Ruimy V, Carvalho AL, Ferreira LMA, Smith SP, Gilbert HJ, Najmudin S, Bayer EA, Fontes CMGA.  2017.  Assembly of Ruminococcus flavefaciens cellulosome revealed by structures of two cohesin-dockerin complexes, 2017. Scientific Reports. 7:759. AbstractWebsite

Cellulosomes are sophisticated multi-enzymatic nanomachines produced by anaerobes to effectively deconstruct plant structural carbohydrates. Cellulosome assembly involves the binding of enzyme-borne dockerins (Doc) to repeated cohesin (Coh) modules located in a non-catalytic scaffoldin. Docs appended to cellulosomal enzymes generally present two similar Coh-binding interfaces supporting a dual-binding mode, which may confer increased positional adjustment of the different complex components. Ruminococcus flavefaciens’ cellulosome is assembled from a repertoire of 223 Doc-containing proteins classified into 6 groups. Recent studies revealed that Docs of groups 3 and 6 are recruited to the cellulosome via a single-binding mode mechanism with an adaptor scaffoldin. To investigate the extent to which the single-binding mode contributes to the assembly of R. flavefaciens cellulosome, the structures of two group 1 Docs bound to Cohs of primary (ScaA) and adaptor (ScaB) scaffoldins were solved. The data revealed that group 1 Docs display a conserved mechanism of Coh recognition involving a single-binding mode. Therefore, in contrast to all cellulosomes described to date, the assembly of R. flavefaciens cellulosome involves single but not dual-binding mode Docs. Thus, this work reveals a novel mechanism of cellulosome assembly and challenges the ubiquitous implication of the dual-binding mode in the acquisition of cellulosome flexibility.

Santos, MFA, Sciortino G, Correia I, Fernandes ACP, Santos-Silva T, Pisanu F, Garribba E, Pessoa JC.  2022.  Binding of VIVO2+, VIVOL, VIVOL2 and VVO2L Moieties to Proteins: X-ray/Theoretical Characterization and Biological Implications, 2022. Chemistry – A European JournalChemistry – A European Journal. 28(40):e202200105.: John Wiley & Sons, Ltd AbstractWebsite

Abstract Vanadium compounds have frequently been proposed as therapeutics, but their application has been hampered by the lack of information on the different V-containing species that may form and how these interact with blood and cell proteins, and with enzymes. Herein, we report several resolved crystal structures of lysozyme with bound VIVO2+ and VIVOL2+, where L=2,2?-bipyridine or 1,10-phenanthroline (phen), and of trypsin with VIVO(picolinato)2 and VVO2(phen)+ moieties. Computational studies complete the refinement and shed light on the relevant role of hydrophobic interactions, hydrogen bonds, and microsolvation in stabilizating the structure. Noteworthy is that the trypsin?VVO2(phen) and trypsin?VIVO(OH)(phen) adducts correspond to similar energies, thus suggesting a possible interconversion under physiological/biological conditions. The obtained data support the relevance of hydrolysis of VIV and VV complexes in the several types of binding established with proteins and the formation of different adducts that might contribute to their pharmacological action, and significantly widen our knowledge of vanadium?protein interactions.

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
n/a
Duarte, RO, Archer M, Dias JM, Bursakov S, Huber R, Moura I, Romao MJ, Moura JJG.  2000.  Biochemical/spectroscopic characterization and preliminary X-ray analysis of a new aldehyde oxidoreductase isolated from Desulfovibrio desulfuricans ATCC 27774. Biochemical and Biophysical Research Communications. 268:745-749., Number 3 AbstractWebsite
n/a
Chaves, S, Gil M, Canario S, Jelic R, Romao MJ, Trincao J, Herdtweck E, Sousa J, Diniz C, Fresco P, Santos AM.  2008.  Biologically relevant O,S-donor compounds. Synthesis, molybdenum complexation and xanthine oxidase inhibition. Dalton Transactions. :1773-1782., Number 13 AbstractWebsite
n/a
Ferreira, P, Cerqueira NSMFA, Fernandes PA, Romão MJ, Ramos MJ.  2020.  Catalytic Mechanism of Human Aldehyde Oxidase, 2020. ACS CatalysisACS Catalysis. 10(16):9276-9286.: American Chemical Society AbstractWebsite

The mechanism of oxidation of N-heterocycle phthalazine to phthalazin-1(2H)-one and its associated free energy profile, catalyzed by human aldehyde oxidase (hAOX1), was studied in atomistic detail using QM/MM methodologies. The studied reaction was found to involve three sequential steps: (i) protonation of the substrate’s N2 atom by Lys893, (ii) nucleophilic attack of the hydroxyl group of the molybdenum cofactor (Moco) to the substrate, and (iii) hydride transfer from the substrate to the sulfur atom of the Moco. The free energy profile that was calculated revealed that the rate-limiting step corresponds to hydride transfer. It was also found that Lys893 plays a relevant role in the reaction, being important not only for the anchorage of the substrate close to the Moco, but also in the catalytic reaction. The variations of the oxidation state of the molybdenum ion throughout the catalytic cycle were examined too. We found out that during the displacement of the products away from the Moco, the transfer of electrons from the catalytic site to the FAD site was proton-coupled. As a consequence, the most favorable and fastest pathway for the enzyme to complete its catalytic cycle was that with MoV and a deprotonated SH ligand of the Moco with the FAD molecule converted to its semiquinone form, FADH•.The mechanism of oxidation of N-heterocycle phthalazine to phthalazin-1(2H)-one and its associated free energy profile, catalyzed by human aldehyde oxidase (hAOX1), was studied in atomistic detail using QM/MM methodologies. The studied reaction was found to involve three sequential steps: (i) protonation of the substrate’s N2 atom by Lys893, (ii) nucleophilic attack of the hydroxyl group of the molybdenum cofactor (Moco) to the substrate, and (iii) hydride transfer from the substrate to the sulfur atom of the Moco. The free energy profile that was calculated revealed that the rate-limiting step corresponds to hydride transfer. It was also found that Lys893 plays a relevant role in the reaction, being important not only for the anchorage of the substrate close to the Moco, but also in the catalytic reaction. The variations of the oxidation state of the molybdenum ion throughout the catalytic cycle were examined too. We found out that during the displacement of the products away from the Moco, the transfer of electrons from the catalytic site to the FAD site was proton-coupled. As a consequence, the most favorable and fastest pathway for the enzyme to complete its catalytic cycle was that with MoV and a deprotonated SH ligand of the Moco with the FAD molecule converted to its semiquinone form, FADH•.

Carvalho, AL, Dias FMV, Prates JAM, Nagy T, Gilbert HJ, Davies GJ, Ferreira LMA, Romao MJ, Fontes C.  2003.  Cellulosome assembly revealed by the crystal structure of the cohesin-dockerin complex. Proceedings of the National Academy of Sciences of the United States of America. 100:13809-13814., Number 24 AbstractWebsite
n/a
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
n/a
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
n/a
Luís, MP, Pereira IS, Bugalhão JN, Simões CN, Mota C, Romão MJ, Mota LJ.  2023.  The Chlamydia trachomatis IncM Protein Interferes with Host Cell Cytokinesis, Centrosome Positioning, and Golgi Distribution and Contributes to the Stability of the Pathogen-Containing Vacuole. Infection and Immunity. 91:e00405-22., Number 4 AbstractWebsite

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes ocular and urogenital infections in humans. The ability of C. trachomatis to grow intracellularly in a pathogen-containing vacuole (known as an inclusion) depends on chlamydial effector proteins transported into the host cell by a type III secretion system. Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes ocular and urogenital infections in humans. The ability of C. trachomatis to grow intracellularly in a pathogen-containing vacuole (known as an inclusion) depends on chlamydial effector proteins transported into the host cell by a type III secretion system. Among these effectors, several inclusion membrane proteins (Incs) insert in the vacuolar membrane. Here, we show that human cell lines infected by a C. trachomatis strain deficient for Inc CT288/CTL0540 (renamed IncM) displayed less multinucleation than when infected by IncM-producing strains (wild type or complemented). This indicated that IncM is involved in the ability of Chlamydia to inhibit host cell cytokinesis. The capacity of IncM to induce multinucleation in infected cells was shown to be conserved among its chlamydial homologues and appeared to require its two larger regions predicted to be exposed to the host cell cytosol. C. trachomatis-infected cells also displayed IncM-dependent defects in centrosome positioning, Golgi distribution around the inclusion, and morphology and stability of the inclusion. The altered morphology of inclusions containing IncM-deficient C. trachomatis was further affected by depolymerization of host cell microtubules. This was not observed after depolymerization of microfilaments, and inclusions containing wild-type C. trachomatis did not alter their morphology upon depolymerization of microtubules. Overall, these findings suggest that IncM may exert its effector function by acting directly or indirectly on host cell microtubules.

Kladova, AV, Gavel YO, Mukhopaadhyay A, Boer DR, Teixeira S, Shnyrov VL, Moura I, Moura JJG, Romao MJ, Trincao J, Bursakov SA.  2009.  Cobalt-, zinc- and iron-bound forms of adenylate kinase (AK) from the sulfate-reducing bacterium Desulfovibrio gigas: purification, crystallization and preliminary X-ray diffraction analysis. Acta Crystallographica Section F-Structural Biology and Crystallization Communications. 65:926-929. AbstractWebsite
n/a