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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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Kumar, K, Correia M, Pires VR, Dhillon A, Sharma K, Rajulapati V, Fontes CMGA, Carvalho AL, Goyal A.  2018.  Novel insights into the degradation of β-1,3-glucans by the cellulosome of Clostridium thermocellum revealed by structure and function studies of a family 81 glycoside hydrolase. International Journal of Biological Macromolecules. :-. AbstractWebsite

Abstract The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a β-1,3-glucanase belonging to cellulosomal complex. The gene encoding \{GH81\} from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of  82 kDa. CtLam81A showed maximum activity against laminarin (100 U/mg), followed by curdlan (65 U/mg), at pH 7.0 and 75 °C. CtLam81A displayed Km, 2.1 ± 0.12 mg/ml and Vmax, 109 ± 1.8 U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca2+ or Mg2+ ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 °C to 96 °C by Ca2+ or Mg2+ ions and decreased to 82 °C by EDTA, indicating that Ca2+ and Mg2+ ions may be involved in catalysis and in maintaining structural integrity. \{TLC\} and MALDI-TOF analysis of β-1,3-glucan hydrolysed products released initially, showed β-1,3-glucan-oligosaccharides degree of polymerization (DP) from \{DP2\} to DP7, confirming an endo-mode of action. The catalytically inactive mutant CtLam81A-E515A generated by site-directed mutagenesis was co-crystallized and tetragonal crystals diffracting up to 1.4 Å resolution were obtained. CtLam81A-E515A contained 15 α-helices and 38 β-strands forming a four-domain structure viz. a β-sandwich domain I at N-terminal, an α/β-domain II, an (α/α)6 barrel domain III, and a small 5-stranded β-sandwich domain IV.

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Boer, DR, Muller A, Fetzner S, Lowe DJ, Romao MJ.  2005.  On the purification and preliminary crystallographic analysis of isoquinoline 1-oxidoreductase from Brevundimonas diminuta 7. Acta Crystallographica Section F-Structural Biology and Crystallization Communications. 61:137-140. AbstractWebsite
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Foti, A, Hartmann T, Coelho C, Santos-Silva T, Romão MJ, Leimkühler S.  2016.  Optimization of the Expression of Human Aldehyde Oxidase for Investigations of Single-Nucleotide Polymorphisms. Drug Metabolism and Disposition. 44:1277–1285., Number 8: American Society for Pharmacology and Experimental Therapeutics AbstractWebsite

Aldehyde oxidase (AOX1) is an enzyme with broad substrate specificity, catalyzing the oxidation of a wide range of endogenous and exogenous aldehydes as well as N-heterocyclic aromatic compounds. In humans, the enzyme’s role in phase I drug metabolism has been established and its importance is now emerging. However, the true physiologic function of AOX1 in mammals is still unknown. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified in human AOX1. SNPs are a major source of interindividual variability in the human population, and SNP-based amino acid exchanges in AOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. For the reliable analysis of the effect of amino acid exchanges in human proteins, the existence of reproducible expression systems for the production of active protein in ample amounts for kinetic, spectroscopic, and crystallographic studies is required. In our study we report an optimized expression system for hAOX1 in Escherichia coli using a codon-optimized construct. The codon-optimization resulted in an up to 15-fold increase of protein production and a simplified purification procedure. The optimized expression system was used to study three SNPs that result in amino acid changes C44W, G1269R, and S1271L. In addition, the crystal structure of the S1271L SNP was solved. We demonstrate that the recombinant enzyme can be used for future studies to exploit the role of AOX in drug metabolism, and for the identification and synthesis of new drugs targeting AOX when combined with crystallographic and modeling studies.

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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Luis, AS, Alves VD, Romao MJ, Prates JAM, Fontes CMGA, Najmudin S.  2011.  Overproduction, purification, crystallization and preliminary X-ray characterization of a novel carbohydrate-binding module of endoglucanase Cel5A from Eubacterium cellulosolvens. Acta Crystallographica Section F-Structural Biology and Crystallization Communications. 67:491-493. AbstractWebsite
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Najmudin, S, Gonzalez PJ, Trincao J, Coelho C, Mukhopadhyay A, Cerqueira NMFSA, Romao CC, Moura I, Moura JJG, Brondino CD, Romao MJ.  2008.  Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum. Journal of Biological Inorganic Chemistry. 13:737-753., Number 5 AbstractWebsite
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Voityuk, AA, Albert K, Kostlmeier S, Nasluzov VA, Neyman KM, Hof P, Huber R, Romao MJ, Rosch N.  1997.  Prediction of alternative structures of the molybdenum site in the xanthine oxidase-related aldehyde oxide reductase. Journal of the American Chemical Society. 119:3159-3160., Number 13 AbstractWebsite
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Romao, MJ, Carvalho AL, Dias JM, Teixeira S, Bourenkov G, Bartunik H, Huber R, Maia L, Mira L.  1999.  Preliminary crystallographic studies of xanthine oxidase purified from rat liver. Journal of Inorganic Biochemistry. 74:281-281., Number 1-4 AbstractWebsite
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Freire, F, Romao MJ, Macedo AL, Aveiro SS, Goodfellow BJ, Carvalho AL.  2009.  Preliminary structural characterization of human SOUL, a haem-binding protein. Acta Crystallographica Section F-Structural Biology and Crystallization Communications. 65:723-726. AbstractWebsite
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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.

Kiefersauer, R, Stetefeld J, GomisRuth FX, Romao MJ, Lottspeich F, Huber R.  1996.  Protein-crystal density by volume measurement and amino-acid analysis. Journal of Applied Crystallography. 29:311-317. AbstractWebsite
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Najmudin, S, Pinheiro BA, Romao MJ, Prates JAM, Fontes CMGA.  2008.  Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-beta-D-xylanase 10B in complex with xylohexaose. Acta Crystallographica Section F-Structural Biology and Crystallization Communications. 64:715-718. AbstractWebsite
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Carvalho, AL, Dias JM, Sanz L, Romero A, Calvete JJ, Romao MJ.  2001.  Purification, crystallization and identification by X-ray analysis of a prostate kallikrein from horse seminal plasma. Acta Crystallographica Section D-Biological Crystallography. 57:1180-1183. AbstractWebsite
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Bras, JLA, Correia MAS, Romao MJ, Prates JAM, Fontes CMGA, Najmudin S.  2011.  Purification, crystallization and preliminary X-ray characterization of the pentamodular arabinoxylanase CtXyl5A from Clostridium thermocellum. Acta Crystallographica Section F-Structural Biology and Crystallization Communications. 67:833-836. AbstractWebsite
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Gavel, OY, Kladova AV, Bursakov SA, Dias JM, Texeira S, Shnyrov VL, Moura JJG, Moura I, Romao MJ, Trincao J.  2008.  Purification, crystallization and preliminary X-ray diffraction analysis of adenosine triphosphate sulfurylase (ATPS) from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774. Acta Crystallographica Section F-Structural Biology and Crystallization Communications. 64:593-595. AbstractWebsite
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Trincao, J, Silva MS, Barata L, Bonifacio C, Carvalho S, Tomas AM, Ferreira AEN, Cordeiro C, Freire AP, Romao MJ.  2006.  Purification, crystallization and preliminary X-ray diffraction analysis of the glyoxalase II from Leishmania infantum. Acta Crystallographica Section F-Structural Biology and Crystallization Communications. 62:805-807. AbstractWebsite
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Najmudin, S, Pinheiro BA, Prates JAM, Gilbert HJ, Romao MJ, Fontes CMGA.  2010.  Putting an N-terminal end to the Clostridium thermocellum xylanase Xyn10B story: Crystal structure of the CBM22-1-GH10 modules complexed with xylohexaose. Journal of Structural Biology. 172:353-362., Number 3 AbstractWebsite
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Viciosa, MT, Correia NT, Salmeron Sanchez M, Carvalho AL, Romao MJ, Gomez Ribelles JL, Dionisio M.  2009.  Real-Time Monitoring of Molecular Dynamics of Ethylene Glycol Dimethacrylate Glass Former. Journal of Physical Chemistry B. 113:14209-14217., Number 43 AbstractWebsite
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dos Santos, R, Carvalho AL, Roque CAA.  2017.  Renaissance of protein crystallization and precipitation in biopharmaceuticals purification, 2017/1//. Biotechnology Advances. 35(1):41-50. AbstractWebsite

AbstractThe current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand. With the upstream improvements, the bottleneck shifted towards the downstream process. New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective. Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules, overcoming the current chromatographic bottlenecks. Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules. This review focuses in the role of these two methodologies in current industrial purification processes, and highlights their potential implementation in the purification pipeline of high value therapeutic molecules, overcoming chromatographic holdups.

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.

Honzicek, J, Mukhopadhyay A, Santos-Silva T, Romao MJ, Romao CC.  2009.  Ring-Functionalized Molybdenocene Complexes. Organometallics. 28:2871-2879., Number 9 AbstractWebsite
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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
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Gomes, AS, Ramos H, Gomes S, Loureiro JB, Soares J, Barcherini V, Monti P, Fronza G, Oliveira C, Domingues L, Bastos M, Dourado DFAR, Carvalho AL, Romão MJ, Pinheiro B, Marcelo F, Carvalho A, Santos MMM, Saraiva L.  2020.  SLMP53-1 interacts with wild-type and mutant p53 DNA-binding domain and reactivates multiple hotspot mutations, 2020. 1864(1):129440. AbstractWebsite

BackgroundHalf of human cancers harbour TP53 mutations that render p53 inactive as a tumor suppressor. As such, reactivation of mutant (mut)p53 through restoration of wild-type (wt)-like function represents one of the most promising therapeutic strategies in cancer treatment. Recently, we have reported the (S)-tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a new reactivator of wt and mutp53 R280K with in vitro and in vivo p53-dependent antitumor activity. The present work aimed a mechanistic elucidation of mutp53 reactivation by SLMP53-1.
Methods and results
By cellular thermal shift assay (CETSA), it is shown that SLMP53-1 induces wt and mutp53 R280K thermal stabilization, which is indicative of intermolecular interactions with these proteins. Accordingly, in silico studies of wt and mutp53 R280K DNA-binding domain with SLMP53-1 unveiled that the compound binds at the interface of the p53 homodimer with the DNA minor groove. Additionally, using yeast and p53-null tumor cells ectopically expressing distinct highly prevalent mutp53, the ability of SLMP53-1 to reactivate multiple mutp53 is evidenced.
Conclusions
SLMP53-1 is a p53-activating agent with the ability to directly target wt and a set of hotspot mutp53.
General Significance
This work reinforces the encouraging application of SLMP53-1 in the personalized treatment of cancer patients harboring distinct p53 status.

Goodfellow, BJ, Tavares P, Romao MJ, Czaja C, Rusnak F, Legall J, Moura I, Moura JJG.  1996.  The solution structure of desulforedoxin, a simple iron-sulfur protein - An NMR study of the zinc derivative. Journal of Biological Inorganic Chemistry. 1:341-354., Number 4 AbstractWebsite
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