Publications

Export 8 results:
Sort by: Author [ Title  (Asc)] Type Year
[A] B C D E F G H I J K L M N O P Q R S T U V W X Y Z   [Show ALL]
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.

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
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.