http://hdl.handle.net/10362/2110 Sun, 19 May 2013 14:20:47 GMT 2013-05-19T14:20:47Z http://run.unl.pt:80/retrieve/57622/bio-chem.jpg http://hdl.handle.net/10362/2110 http://hdl.handle.net/10362/8591 Title: Structure-function relationships in a glycosyltransferase, a phosphatase and an oxidoreductase Authors: Gonçalves, Susana Abstract: Enzyme evolution is often constrained by aspects of catalysis. Mechanistically diverse enzymes evolved from a common ancestor still preserve those structural signatures essential to the core chemistry retained by all members of the superfamily. Indeed, these shared features allow superfamilies to be accurately classified, while derived features allow nested families and subfamilies to be identified in a hierarchical fashion. Accurate classification has helped elucidate mechanisms promoting functional diversification, for example catalytic promiscuity, and protein engineering by rational design. Nowadays, a holistic view of enzymes` regulatory mechanisms and catalytic proficiency is provided by the identification of conserved features of molecular architecture in combination with aspects of reaction dynamics. My work focused on the structural elucidation and analysis of three enzymes: a glycosyltransferase; a phosphatase and an oxidorreductase. “Snapshots” along the reaction coordinate of each enzyme were obtained by combining X-ray diffraction with “cryo-trapping” ligand-binding methods. These were used to characterize the molecular mechanisms involved in substrate recognition and binding. They were also used to distinguish between models proposed for the catalytic mechanisms of each enzyme, and provide insights into enzyme dynamics essential for catalysis and the stereo and regio-selective strategies at work.(...) Description: Dissertation presented to obtain the Ph.D degree in Biochemistry Tue, 01 May 2012 00:00:00 GMT http://hdl.handle.net/10362/8591 2012-05-01T00:00:00Z http://hdl.handle.net/10362/8583 Title: The role of Di-iron proteins in pathogen resistance Authors: Baptista, Joana Morais Abstract: Reactive oxygen and nitrogen species (ROS and RNS) are produced by phagocytic cells of the human immune system to attack invading pathogens due to their ability to damage DNA and the metal centres of proteins. In order to survive inside the host, bacteria activate genes that encode detoxifier enzymes, like the Escherichia coli nitric oxide-reductase flavodiiron protein, also known as flavorubredoxin (FlRd), and repairing proteins, such as the E. coli YtfE di-iron protein involved in the recovery of damaged Fe-S centres. Using E. coli and Staphylococcus aureus, the work presented in this thesis aimed at unravelling: i) the role of E. coli FlRd in bacteria exposed to a combination of oxidative and nitrosative stresses, ii) the identification and characterisation of S. aureus YtfE homologue, iii) the study of E. coli YtfE mechanisms that allow the repair of damaged Fe-S clusters, and iv) the identification of proteins that interact with E. coli YtfE. To analyse the role of E. coli FlRd in cells submitted to both hydrogen peroxide and nitric oxide, the transcription and expression of norV was explored by means of β-galactosidase and immunoblotting assays, respectively. Under these conditions, it was observed that the norV transcription and expression were hindered. To identify if the lack of norV expression was related to its regulator, the NorR transcription factor, the gene was cloned and expressed, and the protein was purified and the binding of nitric oxide to NorR in the presence of hydrogen peroxide was studied. EPR experiments revealed that upon incubation of NorR with nitric oxide and hydrogen peroxide the oxidation promoted by H2O2 of the monoiron centre of NorR impairs the ligation of nitric oxide.(...) Description: Dissertation presented to obtain the Ph.D. degree in Biochemistry Sat, 01 Sep 2012 00:00:00 GMT http://hdl.handle.net/10362/8583 2012-09-01T00:00:00Z http://hdl.handle.net/10362/8562 Title: Insight into the multicopper oxidases stability Authors: Fernandes, André João Tavares Abstract: This dissertation portrays recent development on the knowledge of the stability determinants and of functional characteristics of multicopper oxidases (MCO). Multicopper oxidases are a family of enzymes that includes laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2), ascorbate oxidase (L-ascorbate oxygen oxidoreductase, EC 1.10.3.3) and ceruloplasmin (Fe2+ oxygen oxidoreductase, EC 1.16.3.1). MCO are characterized by having four copper ions that are classified into three distinct types of copper sites, namely type 1 (T1), type 2 (T2) and type 3 (T3). The classical T1 copper site comprises two histidine residues and a cysteine residue arranged in a distorted trigonal geometry around the copper ion with bonding distances approx. 2.0 Å (1 Å=0.1 nm); a weaker fourth methionine ligand completes the tetrahedral geometry. The copper–cysteine linkage is characterized by an intense S(π)→Cu(dx2−y2) CT (charge transfer) absorption band at approximately 600 nm, and a narrow parallel hyperfine splitting A\\ = (43–90)×10−4 cm−1 in the electron paramagnetic resonance (EPR) spectrum. The function of the T1 copper site is to shuttle electrons from substrates to the trinuclear copper centre where molecular oxygen is reduced to two molecules of water during the complete four-electron catalytic cycle. The trinuclear center contains a T2 copper coordinated by two histidine residues and one water molecule, lacks strong absorption bands and exhibits a large parallel hyperfine splitting in the EPR spectrum (A\\ = (150–201)×10−4 cm−1). The T2 copper site is in close proximity to two T3 copper ions, which are each coordinated by three histidine residues and typically coupled, for example, through a dioxygen molecule. The T3 or coupled binuclear copper site is characterized by an intense absorption band at 330 nm originating from the bridging ligand and by the absence of an EPR signal due to the antiferromagnetically coupling of the copper ions.(...) Description: Dissertation presented to obtain the PhD degree in Biochemistry Tue, 01 Feb 2011 00:00:00 GMT http://hdl.handle.net/10362/8562 2011-02-01T00:00:00Z http://hdl.handle.net/10362/8559 Title: Yeast as a model system to study genetic and post-translational regulation of metabolic pathways in mammals Authors: Batista-Nascimento, Liliana Abstract: The work presented in this thesis describes the use of yeast Saccharomyces cerevisiae as a model system to study two different stress response processes and its extrapolation to higher eukaryotes.(...) Description: Dissertation presented to obtain the Ph.D degree in Biology Fri, 01 Jun 2012 00:00:00 GMT http://hdl.handle.net/10362/8559 2012-06-01T00:00:00Z