A carregar...
Projeto de investigação
Detoxification of Hydrogen Peroxide by Pathogenic Bacteria - E.coli Tri-haem peroxidase as a model
Financiador
Autores
Publicações
Biochemical Characterization of the Copper Nitrite Reductase from Neisseria gonorrhoeae
Publication . Barreiro, Daniela S.; Oliveira, Ricardo N. S.; Pauleta, Sofia R.; DQ - Departamento de Química; UCIBIO - Applied Molecular Biosciences Unit; MDPI - Multidisciplinary Digital Publishing Institute
The copper-containing nitrite reductase from Neisseria gonorrhoeae has been shown to play a critical role in the infection mechanism of this microorganism by producing NO and abolishing epithelial exfoliation. This enzyme is a trimer with a type 1 copper center per subunit and a type 2 copper center in the subunits interface, with the latter being the catalytic site. The two centers were characterized for the first time by EPR and CD spectroscopy, showing that the type 1 copper center has a high rhombicity due to its lower symmetry and more tetragonal structure, while the type 2 copper center has the usual properties, but with a smaller hyperfine coupling constant (A// = 10.5 mT). The thermostability of the enzyme was analyzed by differential scanning calorimetry, which shows a single endothermic transition in the thermogram, with a maximum at 94 °C, while the CD spectra in the visible region indicate the presence of the type 1 copper center up to 80 °C. The reoxidation of the N. gonorrhoeae copper-containing nitrite reductase in the presence of nitrite were analyzed by visible spectroscopy and showed a pH dependence, being higher at pH 5.5–6.0. The high thermostability of this enzyme may be important to maintaining a high activity in the extracellular space and to making it less susceptible to denaturation and proteolysis, contributing to the proliferation of N. gonorrhoeae.
Bacterial peroxidases
Publication . Barreiro, Daniela S.; Oliveira, Ricardo N. S.; Pauleta, Sofia R.; DQ - Departamento de Química; UCIBIO - Applied Molecular Biosciences Unit; Elsevier
Bacterial peroxidases are responsible for the reduction of hydrogen peroxide to water. Found in the periplasm of gram-negative bacteria, they are one of the defense mechanisms against endogenous and exogenous peroxide stress under low oxygen tensions. Besides being involved in peroxide detoxification, bacterial peroxidases have been proposed to constitute an alternative pathway to the respiratory chain under anoxic conditions, as demonstrated in E. coli that can use hydrogen peroxide as an electron acceptor in the absence of oxygen. Bacterial peroxidases are c-type cytochromes with either two or three c-type hemes bound to the polypeptide chain, being divided into classical or non-classical, respectively. Orthologous to the classical bacterial peroxidases are the MauG enzymes that share some structural, spectroscopic and sequence similarities but have distinct physiological roles (though for most their function remains unknown). The spectroscopic and kinetic data on bacterial peroxidases are reviewed for both classes. Most classical bacterial peroxidases require reductive activation that consists in structural changes so that the catalytic heme becomes accessible to the substrate. However, non-classical enzymes are ready to bind the hydrogen peroxide as their catalytic center is penta-coordinated, which is also observed in their structural model. The few studies that report the involvement of bacterial peroxidases from pathogenic bacteria in biofilms, is an indication that these enzymes might contribute to their infection mechanism and thus can constitute alternative drug targets
Coordination of the N-Terminal Heme in the Non-Classical Peroxidase from Escherichia coli
Publication . Oliveira, Ricardo N. S.; Aguiar, Sara R. M. M. de; Pauleta, Sofia R.; DQ - Departamento de Química; UCIBIO - Applied Molecular Biosciences Unit; MDPI - Multidisciplinary Digital Publishing Institute
The non-classical bacterial peroxidase from Escherichia coli, YhjA, is proposed to deal with peroxidative stress in the periplasm when the bacterium is exposed to anoxic environments, defending it from hydrogen peroxide and allowing it to thrive under those conditions. This enzyme has a predicted transmembrane helix and is proposed to receive electrons from the quinol pool in an electron transfer pathway involving two hemes (NT and E) to accomplish the reduction of hydrogen peroxide in the periplasm at the third heme (P). Compared with classical bacterial peroxidases, these enzymes have an additional N-terminal domain binding the NT heme. In the absence of a structure of this protein, several residues (M82, M125 and H134) were mutated to identify the axial ligand of the NT heme. Spectroscopic data demonstrate differences only between the YhjA and YhjA M125A variant. In the YhjA M125A variant, the NT heme is high-spin with a lower reduction potential than in the wild-type. Thermostability was studied by circular dichroism, demonstrating that YhjA M125A is thermodynamically more unstable than YhjA, with a lower TM (43 °C vs. 50 °C). These data also corroborate the structural model of this enzyme. The axial ligand of the NT heme was validated to be M125, and mutation of this residue was proven to affect the spectroscopic, kinetic, and thermodynamic properties of YhjA.
Structural Characterization of Neisseria gonorrhoeae Bacterial Peroxidase—Insights into the Catalytic Cycle of Bacterial Peroxidases
Publication . Nóbrega, Cláudia S.; Carvalho, Ana Luísa; Romão, Maria João; Pauleta, Sofia R.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; MDPI - Multidisciplinary Digital Publishing Institute
Neisseria 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.
Unidades organizacionais
Descrição
Palavras-chave
Contribuidores
Financiadores
Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
Concurso para Financiamento de Projetos de Investigação Científica e Desenvolvimento Tecnológico em Todos os Domínios Científicos - 2017
Número da atribuição
PTDC/BIA-BQM/29442/2017