Coelho, C, Romao MJ.
2015.
Structural and mechanistic insights on nitrate reductases, 2015. Protein Science. 24(12):1901-1911.
AbstractNitrate reductases (NR) belong to the DMSO reductase family of Mo-containing enzymes and perform key roles in the metabolism of the nitrogen cycle, reducing nitrate to nitrite. Due to variable cell location, structure and function, they have been divided into periplasmic (Nap), cytoplasmic, and membrane-bound (Nar) nitrate reductases. The first crystal structure obtained for a NR was that of the monomeric NapA from Desulfovibrio desulfuricans in 1999. Since then several new crystal structures were solved providing novel insights that led to the revision of the commonly accepted reaction mechanism for periplasmic nitrate reductases. The two crystal structures available for the NarGHI protein are from the same organism (Escherichia coli) and the combination with electrochemical and spectroscopic studies also lead to the proposal of a reaction mechanism for this group of enzymes. Here we present an overview on the current advances in structural and functional aspects of bacterial nitrate reductases, focusing on the mechanistic implications drawn from the crystallographic data.
Romão, MJ, Coelho C, Santos-Silva T, Foti A, Terao M, Garattini E, Leimkühler S.
2017.
Structural basis for the role of mammalian aldehyde oxidases in the metabolism of drugs and xenobiotics. Current Opinion in Chemical Biology. 37:39-47.
AbstractAldehyde oxidases (AOXs) are molybdo-flavoenzymes characterized by broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into the corresponding carboxylic acids and hydroxylating various heteroaromatic rings. Mammals are characterized by a complement of species-specific \{AOX\} isoenzymes, that varies from one in humans (AOX1) to four in rodents (AOX1, AOX2, \{AOX3\} and AOX4). The physiological function of mammalian \{AOX\} isoenzymes is unknown, although human \{AOX1\} is an emerging enzyme in phase-I drug metabolism. Indeed, the number of therapeutic molecules under development which act as \{AOX\} substrates is increasing. The recent crystallization and structure determination of human \{AOX1\} as well as mouse \{AOX3\} has brought new insights into the mechanisms underlying substrate/inhibitor binding as well as the catalytic activity of this class of enzymes.
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
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Nóbrega, CS, Carvalho AL, Romão MJ, Pauleta SR.
2023.
Structural Characterization of Neisseria gonorrhoeae Bacterial Peroxidase—Insights into the Catalytic Cycle of Bacterial Peroxidases. International Journal of Molecular Sciences. 24, Number 7
AbstractNeisseria gonorrhoeae is an obligate human pathogenic bacterium responsible for gonorrhea, a sexually transmitted disease. The bacterial peroxidase, an enzyme present in the periplasm of this bacterium, detoxifies the cells against hydrogen peroxide and constitutes one of the primary defenses against exogenous and endogenous oxidative stress in this organism. The 38 kDa heterologously produced bacterial peroxidase was crystallized in the mixed-valence state, the active state, at pH 6.0, and the crystals were soaked with azide, producing the first azide-inhibited structure of this family of enzymes. The enzyme binds exogenous ligands such as cyanide and azide, which also inhibit the catalytic activity by coordinating the P heme iron, the active site, and competing with its substrate, hydrogen peroxide. The inhibition constants were estimated to be 0.4 ± 0.1 µM and 41 ± 5 mM for cyanide and azide, respectively. Imidazole also binds and inhibits the enzyme in a more complex mechanism by binding to P and E hemes, which changes the reduction potential of the latest heme. Based on the structures now reported, the catalytic cycle of bacterial peroxidases is revisited. The inhibition studies and the crystal structure of the inhibited enzyme comprise the first platform to search and develop inhibitors that target this enzyme as a possible new strategy against N. gonorrhoeae.
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
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Lima, CDL, Coelho H, Gimeno A, Trovão F, Diniz A, Dias JS, Jiménez-Barbero J, Corzana F, Carvalho AL, Cabrita EJ, Marcelo F.
2021.
Structural insights into the molecular recognition mechanism of the cancer and pathogenic epitope, LacdiNAc by immune-related lectins, 2021. Chemistry – A European JournalChemistry – A European Journal. n/a(n/a): John Wiley & Sons, Ltd
AbstractInteractions of glycan-specific epitopes to human lectin receptors represent novel immune checkpoints for investigating cancer and infection diseases. By employing a multidisciplinary approach that combines isothermal titration calorimetry, NMR spectroscopy, molecular dynamics simulations, and X-ray crystallography, we disclosed the molecular determinants that govern the recognition of the tumour and pathogenic glycobiomarker LacdiNAc (GalNAc?1-4GlcNAc, LDN), including their comparison with the ubiquitous LacNAc epitope (Gal?1-4GlcNAc, LN), by two human immune-related lectins, galectin-3 (hGal-3) and the macrophage galactose C-type lectin (hMGL). A different mechanism of binding and interactions is observed for the hGal-3/LDN and hMGL/LDN complexes, which explains the remarkable difference in the binding specificity of LDN and LN by these two lectins. The new structural clues reported herein are fundamental for the chemical design of mimetics targeting hGal-3/hMGL recognition process.
Goncalves, LML, Cunha C, Almeida G, Macieira S, Costa C, Lampreia J, Romao MJ, Moura JJG, Moura I.
2001.
Structural studies on Desulfovibrio desulfuricans ATCC 27774 multiheme nitrite reductase - characterization of the subunits. Journal of Inorganic Biochemistry. 86:316-316., Number 1
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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
AbstractMammalian 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.
Huber, R, Hof P, Duarte RO, Moura JJG, Moura I, Liu MY, Legall J, Hille R, Archer M, Romao MJ.
1996.
A structure-based catalytic mechanism for the xanthine oxidase family of molybdenum enzymes. Proceedings of the National Academy of Sciences of the United States of America. 93:8846-8851., Number 17
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Vilela-Alves, G, Manuel RR, Viegas A, Carpentier P, Biaso F, Guigliarelli B, Pereira IAC, Romão MJ, Mota C.
2024.
Substrate-dependent oxidative inactivation of a W-dependent formate dehydrogenase involving selenocysteine displacement. bioRxiv. : Cold Spring Harbor Laboratory
AbstractMetal-dependent formate dehydrogenases are very promising targets for enzyme optimization and design of bio-inspired catalysts for CO2 reduction, towards novel strategies for climate change mitigation. For effective application of these enzymes, the catalytic mechanism must be fully understood, and the molecular determinants clarified. Despite numerous studies, several doubts persist, namely regarding the role played by the possible dissociation of the SeCys ligand from the Mo/W active site. Additionally, the O2 sensitivity of these enzymes must also be understood as it poses an important obstacle for biotechnological applications. Here we present a combined biochemical, spectroscopic, and structural characterization of Desulfovibrio vulgaris FdhAB (DvFdhAB) when exposed to oxygen in the presence of a substrate (formate or CO2). This study reveals that O2 inactivation is promoted by the presence of either substrate and involves forming a new species in the active site, captured in the crystal structures, where the SeCys ligand is displaced from tungsten coordination and replaced by a dioxygen or peroxide molecule. This new form was reproducibly obtained and supports the conclusion that, although W-DvFdhAB can catalyze the oxidation of formate in the presence of oxygen for some minutes, it gets irreversibly inactivated after prolonged O2 exposure in the presence of either substrate. These results reveal that oxidative inactivation does not require reduction of the metal, as widely assumed, as it can also occur in the oxidized state in the presence of CO2.Competing Interest StatementThe authors have declared no competing interest.AORAldehyde Oxido-reductaseDTTDithiothreitolDvDesulfovibrio vulgarisEPRElectron Paramagnetic ResonanceFdhFormate dehydrogenaseHPHigh PressureMGDMolybdopterin Guanine DinucleotidesNDNew dropROSReactive Oxygen SpeciesSODSuperoxide dismutaseTSAThermal Shift Assay