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Ítem Acceso Abierto Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: simulation, kinetics and evolutionary studies(Public Library of Science (PLOS), 2016-03-24) Fuentealba, Matías; Muñoz, Rodrigo; Maturana, Pablo; Krapp, Adriana R.; Cabrera, RicardoGlucose 6-Phosphate Dehydrogenases (G6PDHs) from different sources show varying specificities towards NAD+ and NADP+ as cofactors. However, it is not known to what extent structural determinants of cofactor preference are conserved in the G6PDH family. In this work, molecular simulations, kinetic characterization of site-directed mutants and phylogenetic analyses were used to study the structural basis for the strong preference towards NADP+ shown by the G6PDH from Escherichia coli. Molecular Dynamics trajectories of homology models showed a highly favorable binding energy for residues K18 and R50 when interacting with the 2'-phosphate of NADP+, but the same residues formed no observable interactions in the case of NAD+. Alanine mutants of both residues were kinetically characterized and analyzed with respect to the binding energy of the transition state, according to the kcat/KM value determined for each cofactor. Whereas both residues contribute to the binding energy of NADP+, only R50 makes a contribution (about -1 kcal/mol) to NAD+ binding. In the absence of both positive charges the enzyme was unable to discriminate NADP+ from NAD+. Although kinetic data is sparse, the observed distribution of cofactor preferences within the phylogenetic tree is sufficient to rule out the possibility that the known NADP+-specific G6PDHs form a monophyletic group. While the β1-α1 loop shows no strict conservation of K18, (rather, S and T seem to be more frequent), in the case of the β2-α2 loop, different degrees of conservation are observed for R50. Noteworthy is the fact that a K18T mutant is indistinguishable from K18A in terms of cofactor preference. We conclude that the structural determinants for the strict discrimination against NAD+ in the case of the NADP+-specific enzymes have evolved independently through different means during the evolution of the G6PDH family. We further suggest that other regions in the cofactor binding pocket, besides the β1-α1 and β2-α2 loops, play a role in determining cofactor preference.Ítem Acceso Abierto A long-chain flavodoxin protects pseudomonas aeruginosa from oxidative stress and host bacterial clearance(Public Library of Science (PLOS), 2014-02-13) Moyano, Alejandro José; Tobares, Romina Alín; Rizzi, Yanina S.; Krapp, Adriana R.; Mondotte, Juan Alberto; Bocco, José Luis; Saleh, Maria-Carla; Carrillo, Néstor; Smania, Andrea M.Long-chain flavodoxins, ubiquitous electron shuttles containing flavin mononucleotide (FMN) as prosthetic group, play an important protective role against reactive oxygen species (ROS) in various microorganisms. Pseudomonas aeruginosa is an opportunistic pathogen which frequently has to face ROS toxicity in the environment as well as within the host. We identified a single ORF, hereafter referred to as fldP (for flavodoxin from P. aeruginosa), displaying the highest similarity in length, sequence identity and predicted secondary structure with typical long-chain flavodoxins. The gene was cloned and expressed in Escherichia coli. The recombinant product (FldP) could bind FMN and exhibited flavodoxin activity in vitro. Expression of fldP in P. aeruginosa was induced by oxidative stress conditions through an OxyR-independent mechanism, and an fldP-null mutant accumulated higher intracellular ROS levels and exhibited decreased tolerance to H2O2 toxicity compared to wild-type siblings. The mutant phenotype could be complemented by expression of a cyanobacterial flavodoxin. Overexpression of FldP in a mutT-deficient P. aeruginosa strain decreased H2O2-induced cell death and the hypermutability caused by DNA oxidative damage. FldP contributed to the survival of P. aeruginosa within cultured mammalian macrophages and in infected Drosophila melanogaster, which led in turn to accelerated death of the flies. Interestingly, the fldP gene is present in some but not all P. aeruginosa strains, constituting a component of the P. aeruginosa accessory genome. It is located in a genomic island as part of a self-regulated polycistronic operon containing a suite of stress-associated genes. The collected results indicate that the fldP gene encodes a long-chain flavodoxin, which protects the cell from oxidative stress, thereby expanding the capabilities of P. aeruginosa to thrive in hostile environments.Ítem Acceso Abierto Transcriptional and metabolic changes associated to the infection by fusarium verticillioides in maize inbreds with contrasting ear rot resistance(Public Library of Science (PLOS), 2013-04-18) Campos Bermudez, Valeria A.; Fauguel, Carolina M.; Tronconi, Marcos A.; Casati, Paula; Presello, Daniel A.; Andreo, Carlos S.Fusarium verticillioides causes ear rot and grain mycotoxins in maize (Zea mays L.), which are harmful to human and animal health. Breeding and growing less susceptible plant genotypes is one alternative to reduce these detrimental effects. A better understanding of the resistance mechanisms would facilitate the implementation of strategic molecular agriculture to breeding of resistant germplasm. Our aim was to identify genes and metabolites that may be related to the Fusarium reaction in a resistant (L4637) and a susceptible (L4674) inbred. Gene expression data were obtained from microarray hybridizations in inoculated and non-inoculated kernels from both inbreds. Fungal inoculation did not produce considerable changes in gene expression and metabolites in L4637. Defense-related genes changed in L4674 kernels, responding specifically to the pathogen infection. These results indicate that L4637 resistance may be mainly due to constitutive defense mechanisms preventing fungal infection. These mechanisms seem to be poorly expressed in L4674; and despite the inoculation activate a defense response; this is not enough to prevent the disease progress in this susceptible line. Through this study, a global view of differential genes expressed and metabolites accumulated during resistance and susceptibility to F. verticillioides inoculation has been obtained, giving additional information about the mechanisms and pathways conferring resistance to this important disease in maize.Ítem Acceso Abierto Engineering Climate-Change-Resilient Crops: New Tools and Approaches(MDPI, 2021-07-23) Shahinnia, Fahimeh; Carrillo, Néstor; Hajirezaei, Mohammad-RezaÍtem Acceso Abierto CuA-based chimeric T1 copper sites allow for independent modulation of reorganization energy and reduction potential(Royal Society of Chemistry, 2021-02-04) Szuster, Jonathan; Zitare, Ulises A.; Castro, María A.; Leguto, Alcides J.; Morgada, Marcos Nicolás; Vila, Alejandro J.; Murgida, Daniel H.Ítem Acceso Abierto Environmental selection pressures related to iron utilization are involved in the loss of the flavodoxin gene from the plant genome(Oxford University Press, 2015-02-16) Pierella Karlusich, Juan J.; Ceccoli, Romina Denis; Graña, Martín; Romero, Héctor; Carrillo, NéstorÍtem Acceso Abierto The heme uptake process in Trypanosoma cruzi epimastigotes is inhibited by heme analogues and by inhibitors of ABC transporters(Elsevier, 2011) Cupello Peixoto, Mauricio; Fernandes de Souza, Cíntia; Buchensky, Celeste; Rocha Corrêa Soares, Juliana Baptista; Travassos Laranja, Gustavo Augusto; Garcia Pinto Coelho, Marsen; Cricco, Julia Alejandra; Paes, Marcia CristinaÍtem Acceso Abierto Heme A synthesis and CcO activity are essential for Trypanosoma cruzi infectivity and replication(Portland Press, 2017-06-27) Merli, Marcelo Luciano; Cirulli, Brenda Analía; Menéndez-Bravo, Simón M.; Cricco, Julia AlejandraÍtem Acceso Abierto Biosynthesis of heme O in intraerythrocytic stages of Plasmodium falciparum and potential inhibitors of this pathway(Springer Nature, 2019-12-17) Simão-Gurge, Raquel M.; Wunderlich, Gerhard; Cricco, Julia Alejandra; Galindo Cubillos, Eliana F.; Domenech-Carbó, Antonio; Cebrián-Torrejón, Gerardo; Almeida, Fernando G.; Cirulli, Brenda Analía; Katzin, Alejandro M.Ítem Acceso Abierto The Trypanosoma cruzi Protein TcHTE Is Critical for Heme Uptake(Public Library of Science PLOS, 2016-01) Merli, Marcelo Luciano; Pagura, Lucas; Hernández, Josefina; Barisón, María Julia; Pral, Elizabeth M. F.; Silber, Ariel M.; Cricco, Julia AlejandraÍtem Acceso Abierto Role of Heme and Heme-Proteins in Trypanosomatid essential metabolic pathways(Hindawi, 2011) Tripodi, Karina E. J.; Menendez Bravo, Simón M.; Cricco, Julia AlejandraÍtem Acceso Abierto The Trypanosoma cruzi proteins TcCox10 and TcCox15 catalyze the formation of heme A in the yeast Saccharomyces cerevisiae(Oxford University Press, 2010-11) Buchensky, Celeste; Almirón, Paula; Suarez Mantilla, Brian; Silber, Ariel M.; Cricco, Julia Alejandra