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Oeiras, Portugal

Utesch T.,TU Berlin | Sezer M.,ITQB UNL | Weidinger I.M.,TU Berlin | Mroginski M.A.,TU Berlin
Langmuir | Year: 2012

Sulfite oxidase (SO) is an enzyme catalyzing the terminal step of the metabolism of sulfur-containing amino acids that is essential for almost all living organisms. The catalytic activity of SO in vertebrates strongly depends on the efficiency of the intramolecular electron transfer (IET) between the catalytic Moco domain and the cytochrome b5 (cyt b5) domain. The IET process is assumed to be mediated by large domain motions of the cyt b5 domains within the enzyme. Thus, the interaction of SO with charged surfaces may affect the mobility of the cyt b5 domain required for IET and consequently hinder SO activation. In this study, we present a molecular dynamics approach to investigating the ionic strength dependence of the initial surface adsorption of SO in two different conformations - the crystallographic structure and the model structure for an activated SO - onto mixed amino- and hydroxyl-terminated SAMs. The results show for both conformations at low ionic strengths a strong adsorption of the cyt b5 units onto the SAM, which inhibits the domain motion event required for IET. Under higher ion concentrations, however, the interaction with the surface is weakened by the negatively charged ions acting as a buffer and competing in adsorption with the cathodic cyt b5 domains. This competition prevents the immobilization of the cytochrome b5 units onto the surface, allowing the intramolecular domain motions favoring IET. Our predictions support the interpretation of recent experimental spectroelectrochemical studies on SO. © 2012 American Chemical Society. Source

Costa R.S.,Institute Engineering Of Sistemas E Computadores | Hartmann A.,Institute Engineering Of Sistemas E Computadores | Gaspar P.,ITQB UNL | Neves A.R.,ITQB UNL | And 2 more authors.
Molecular BioSystems | Year: 2014

Biomedical research and biotechnological production are greatly benefiting from the results provided by the development of dynamic models of microbial metabolism. Although several kinetic models of Lactococcus lactis (a Lactic Acid Bacterium (LAB) commonly used in the dairy industry) have been developed so far, most of them are simplified and focus only on specific metabolic pathways. Therefore, the application of mathematical models in the design of an engineering strategy for the production of industrially important products by L. lactis has been very limited. In this work, we extend the existing kinetic model of L. lactis central metabolism to include industrially relevant production pathways such as mannitol and 2,3-butanediol. In this way, we expect to study the dynamics of metabolite production and make predictive simulations in L. lactis. We used a system of ordinary differential equations (ODEs) with approximate Michaelis-Menten-like kinetics for each reaction, where the parameters were estimated from multivariate time-series metabolite concentrations obtained by our team through in vivo Nuclear Magnetic Resonance (NMR). The results show that the model captures observed transient dynamics when validated under a wide range of experimental conditions. Furthermore, we analyzed the model using global perturbations, which corroborate experimental evidence about metabolic responses upon enzymatic changes. These include that mannitol production is very sensitive to lactate dehydrogenase (LDH) in the wild type (W.T.) strain, and to mannitol phosphoenolpyruvate: a phosphotransferase system (PTSMtl) in a LDH mutant strain. LDH reduction has also a positive control on 2,3-butanediol levels. Furthermore, it was found that overproduction of mannitol-1-phosphate dehydrogenase (MPD) in a LDH/PTS Mtl deficient strain can increase the mannitol levels. The results show that this model has prediction capability over new experimental conditions and offers promising possibilities to elucidate the effect of alterations in the main metabolism of L. lactis, with application in strain optimization. © 2014 The Royal Society of Chemistry. Source

Vicari C.,University of Naples Federico II | Saraiva I.H.,ITQB UNL | Maglio O.,University of Naples Federico II | Maglio O.,CNR Institute of Biostructure and Bioimaging | And 4 more authors.
Chemical Communications | Year: 2014

An empirical equation, describing the relationship between the porphyrin methyl hyperfine shifts and the position of the axial ligand(s), has been applied to an artificial heme-protein in order to obtain insight into the active site properties of heme-protein models. This journal is © the Partner Organisations 2014. Source

Cal P.M.S.D.,University of Lisbon | Vicente J.B.,University of Lisbon | Pires E.,ITQB UNL | Coelho A.V.,ITQB UNL | And 3 more authors.
Journal of the American Chemical Society | Year: 2012

Protein modification has entered the limelight of chemical and biological sciences, since, by appending small molecules into proteins surfaces, fundamental biological and biophysical processes may be studied and even modulated in a physiological context. Herein we present a new strategy to modify the lysines η-amino group and the proteins N-terminal, based on the formation of stable iminoboronates in aqueous media. This functionality enables the stable and complete modification of these amine groups, which can be reversible upon the addition of fructose, dopamine, or glutathione. A detailed DFT study is also presented to rationalize the observed stability toward hydrolysis of the iminoboronate constructs. © 2012 American Chemical Society. Source

Paquete C.M.,ITQB UNL | Louro R.O.,ITQB UNL
Dalton Transactions | Year: 2010

Shewanella are facultative anaerobic bacteria of remarkable respiratory versatility that includes the dissimilatory reduction of metal ores. They contain a large number of multiheme c-type cytochromes that play a significant role in various anaerobic respiratory processes. Of all the cytochromes found in Shewanella, only the two most abundant periplasmic cytochromes, the small tetraheme cytochrome (STC) and flavocytochrome c3 (Fcc3) have been structurally characterized. For these two proteins the molecular bases for their redox properties were determined using spectroscopic methods based on paramagnetic NMR, that allow the contribution of specific hemes to be discriminated. In this perspective these results are reviewed in the context of the continuing effort to understand the molecular mechanisms of electron transfer in the respiratory chains of these organisms. © 2010 The Royal Society of Chemistry. Source

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