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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
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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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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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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
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Brás, NF, Neves RPP, Lopes FAA, Correia MAS, Palma AS, Sousa SF, Ramos MJ.  2021.  Combined in silico and in vitro studies to identify novel antidiabetic flavonoids targeting glycogen phosphorylase, 2021. 108:104552. AbstractWebsite

Novel pharmacological strategies for the treatment of diabetic patients are now focusing on inhibiting glycogenolysis steps. In this regard, glycogen phosphorylase (GP) is a validated target for the discovery of innovative antihyperglycemic molecules. Natural products, and in particular flavonoids, have been reported as potent inhibitors of GP at the cellular level. Herein, free-energy calculations and microscale thermophoresis approaches were performed to get an in-depth assessment of the binding affinities and elucidate intermolecular interactions of several flavonoids at the inhibitor site of GP. To our knowledge, this is the first study indicating genistein, 8-prenylgenistein, apigenin, 8-prenylapigenin, 8-prenylnaringenin, galangin and valoneic acid dilactone as natural molecules with high inhibitory potency toward GP. We identified: i) the residues Phe285, Tyr613, Glu382 and/or Arg770 as the most relevant for the binding of the best flavonoids to the inhibitor site of GP, and ii) the 5-OH, 7-OH, 8-prenyl substitutions in ring A and the 4′-OH insertion in ring B to favor flavonoid binding at this site. Our results are invaluable to plan further structural modifications through organic synthesis approaches and develop more effective pharmaceuticals for Type 2 Diabetes treatment, and serve as the starting point for the exploration of food products for therapeutic usage, as well as for the development of novel bio-functional food and dietary supplements/herbal medicines.

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.

Santos-Silva, T, Mukhopadhyay A, Seixas JD, Bernardes GJL, Romao CC, Romao MJ.  2011.  CORM-3 Reactivity toward Proteins: The Crystal Structure of a Ru(II) Dicarbonyl-Lysozyme Complex. Journal of the American Chemical Society. 133:1192-1195., Number 5 AbstractWebsite
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Thapper, A, Boer DR, Brondino CD, Moura JJG, Romao MJ.  2007.  Correlating EPR and X-ray structural analysis of arsenite-inhibited forms of aldehyde oxidoreductase. Journal of Biological Inorganic Chemistry. 12:353-366., Number 3 AbstractWebsite
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Mota, C, Coelho C, Leimkühler S, Garattini E, Terao M, Santos-Silva T, Romão MJ.  2018.  Critical overview on the structure and metabolism of human aldehyde oxidase and its role in pharmacokinetics, 2018. 368:35-59. AbstractWebsite

Aldehyde oxidases are molybdenum and flavin dependent enzymes characterized by a very wide substrate specificity and performing diverse reactions that include oxidations (e.g., aldehydes and aza-heterocycles), hydrolysis of amide bonds, and reductions (e.g., nitro, S-oxides and N-oxides). Oxidation reactions and amide hydrolysis occur at the molybdenum site while the reductions are proposed to occur at the flavin site. AOX activity affects the metabolism of different drugs and xenobiotics, some of which designed to resist other liver metabolizing enzymes (e.g., cytochrome P450 monooxygenase isoenzymes), raising its importance in drug development. This work consists of a comprehensive overview on aldehyde oxidases, concerning the genetic evolution of AOX, its diversity among the human population, the crystal structures available, the known catalytic reactions and the consequences in pre-clinical pharmacokinetic and pharmacodynamic studies. Analysis of the different animal models generally used for pre-clinical trials and comparison between the human (hAOX1), mouse homologs as well as the related xanthine oxidase (XOR) are extensively considered. The data reviewed also include a systematic analysis of representative classes of molecules that are hAOX1 substrates as well as of typical and well characterized hAOX1 inhibitors. The considerations made on the basis of a structural and functional analysis are correlated with reported kinetic and metabolic data for typical classes of drugs, searching for potential structural determinants that may dictate substrate and/or inhibitor specificities.

Bursakov, SA, Brondino C, Dias JM, Carneiro C, Caldeira J, Duarte RO, Romao MJ, Moura I, Moura JJG.  1999.  Cross immunological reactions and spectroscopy study within nitrate reductase and other mononuclear Mo containing enzymes of the sulfate reducing bacteria. Journal of Inorganic Biochemistry. 74:86-86., Number 1-4 AbstractWebsite
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Romao, MJ, Hubert R.  1997.  Crystal structure and mechanism of action of the xanthine oxidase-related aldehyde oxidoreductase from Desulfovibrio gigas. Biochemical Society Transactions. 25:755-757., Number 3 AbstractWebsite
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Carvalho, AL, Sanz L, Barettino D, Romero A, Calvete JJ, Romao MJ.  2002.  Crystal structure of a prostate kallikrein isolated from stallion seminal plasma: A homologue of human PSA. Journal of Molecular Biology. 322:325-337., Number 2 AbstractWebsite
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Romao, MJ, Kolln I, Dias JM, Carvalho AL, Romero A, Varela PF, Sanz L, Topfer-Petersen E, Calvete JJ.  1997.  Crystal structure of acidic seminal fluid protein (aSFP) at 1.9 angstrom resolution: a bovine polypeptide of the spermadhesin family. Journal of Molecular Biology. 274:650-660., Number 4 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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Archer, M, Banci L, Dikaya E, Romao MJ.  1997.  Crystal structure of cytochrome c' from Rhodocyclus gelatinosus and comparison with other cytochromes c'. Journal of Biological Inorganic Chemistry. 2:611-622., Number 5 AbstractWebsite
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Romero, A, Caldeira J, Legall J, Moura I, Moura JJG, Romao MJ.  1996.  Crystal structure of flavodoxin from Desulfovibrio desulfuricans ATCC 27774 in two oxidation states. European Journal of Biochemistry. 239:190-196., Number 1 AbstractWebsite
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Santos-Silva, T, Dias JM, Dolla A, Durand M-C, Goncalves LL, Lampreia J, Moura I, Romao MJ.  2007.  Crystal structure of the 16 heme cytochrome from Desulfovibrio gigas: A glycosylated protein in a sulphate-reducing bacterium. Journal of Molecular Biology. 370:659-673., Number 4 AbstractWebsite
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Dias, JM, Than ME, Humm A, Huber R, Bourenkov GP, Bartunik HD, Bursakov S, Calvete J, Caldeira J, Carneiro C, Moura JJG, Moura I, Romao MJ.  1999.  Crystal structure of the first dissimilatory nitrate reductase at 1.9 angstrom solved by MAD methods. Structure with Folding & Design. 7:65-79., Number 1 AbstractWebsite
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Gomes, AS, Trovão F, Andrade Pinheiro B, Freire F, Gomes S, Oliveira C, Domingues L, Romão MJ, Saraiva L, Carvalho AL.  2018.  The Crystal Structure of the R280K Mutant of Human p53 Explains the Loss of DNA Binding. International Journal of Molecular Sciences. 19, Number 4}, ARTICLE NUMBER = {1184 AbstractWebsite

The p53 tumor suppressor is widely found to be mutated in human cancer. This protein is regarded as a molecular hub regulating different cell responses, namely cell death. Compelling data have demonstrated that the impairment of p53 activity correlates with tumor development and maintenance. For these reasons, the reactivation of p53 function is regarded as a promising strategy to halt cancer. In the present work, the recombinant mutant p53R280K DNA binding domain (DBD) was produced for the first time, and its crystal structure was determined in the absence of DNA to a resolution of 2.0 Å. The solved structure contains four molecules in the asymmetric unit, four zinc(II) ions, and 336 water molecules. The structure was compared with the wild-type p53 DBD structure, isolated and in complex with DNA. These comparisons contributed to a deeper understanding of the mutant p53R280K structure, as well as the loss of DNA binding related to halted transcriptional activity. The structural information derived may also contribute to the rational design of mutant p53 reactivating molecules with potential application in cancer treatment.

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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Romero, A, Romao MJ, Varela PF, Kolln I, Dias JM, Carvalho AL, Sanz L, TopferPetersen E, Calvete JJ.  1997.  The crystal structures of two spermadhesins reveal the CUB domain fold. Nature Structural Biology. 4:783-788., Number 10 AbstractWebsite
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Romao, MJ, Turk D, GomisRuth FX, Huber R, Schumacher G, Mollering H, Russmann L.  1992.  CRYSTAL-STRUCTURE ANALYSIS, REFINEMENT AND ENZYMATIC-REACTION MECHANISM OF N-CARBAMOYLSARCOSINE AMIDOHYDROLASE FROM ARTHROBACTER SP AT 2.0-ANGSTROM RESOLUTION. Journal of Molecular Biology. 226:1111-1130., Number 4 AbstractWebsite
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Archer, M, Huber R, Tavares P, Moura I, Moura JJG, Carrondo MA, Sieker LC, Legall J, Romao MJ.  1995.  CRYSTAL-STRUCTURE OF DESULFOREDOXIN FROM DESULFOVIBRIO-GIGAS DETERMINED AT 1.8 ANGSTROM RESOLUTION - A NOVEL NONHEME IRON PROTEIN-STRUCTURE. Journal of Molecular Biology. 251:690-702., Number 5 AbstractWebsite
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