A Highly Stable Plastidic-Type Ferredoxin-NADP(H) Reductase in the Pathogenic Bacterium Leptospira interrogans

Leptospira interrogans is a bacterium that is capable of infecting animals and humans, and its infection causes leptospirosis with a range of symptoms from flu-like to severe illness and death. Despite being a bacteria, Leptospira interrogans contains a plastidic class ferredoxin-NADP(H) reductase (FNR) with high catalytic efficiency, at difference from the bacterial class FNRs. These flavoenzymes catalyze the electron transfer between NADP(H) and ferredoxins or flavodoxins. The inclusion of a plastidic FNR in Leptospira metabolism and in its parasitic life cycle is not currently understood. Bioinformatic analyses of the available genomic and proteins sequences showed that the presence of this enzyme in nonphotosynthetic bacteria is restricted to the Leptospira genus and that a [4Fe-4S] ferredoxin (LB107) encoded by the Leptospira genome may be the natural substrate of the enzyme. Leptospira FNR (LepFNR) displayed high diaphorase activity using artificial acceptors and functioned as a ferric reductase. LepFNR displayed cytochrome c reductase activity with the Leptospira LB107 ferredoxin with an optimum at pH 6.5. Structural stability analysis demonstrates that LepFNR is one of the most stable FNRs analyzed to date. The persistence of a native folded LepFNR structure was detected in up to 6 M urea, a condition in which the enzyme retains 38% activity. In silico analysis indicates that the high LepFNR stability might be due to robust interactions between the FAD and the NADP+ domains of the protein. The limited bacterial distribution of plastidic class FNRs and the biochemical and structural properties of LepFNR emphasize the uniqueness of this enzyme in the Leptospira metabolism. Our studies show that in L. interrogans a plastidic-type FNR exchanges electrons with a bacterial-type ferredoxin, process which has not been previously observed in nature

Potencialidades de la enzima L-glutaminasa en la industria de alimentos

Las enzimas representan una herramienta de gran potencial para abordar las demandas de la industria de alimentos y su manufactura ha emergido como una de las industrias estratégicas para generar valor agregado. La enzima L-glutaminasa cataliza la desaminación hidrolítica del aminoácido L-glutamina, produciendo ácido L-glutámico, un potenciador del sabor, y amonio, un neutralizador de la acidez. Los hidrolizados proteicos presentan distintas actividades biológicas beneficiosas para la salud. Sin embargo, el sabor amargo característico es el principal factor que limita su rango de aplicaciones. Los métodos fisicoquímicos frecuentemente usados para disminuir el sabor amargo ocasionan resultados no deseados. Para superar esta limitación la modificación inducida por desaminación con L-glutaminasa ha sido propuesta como una técnica efectiva para mejorar el sabor. Este proceso además aumenta la solubilidad de hidrolizados proteicos y mejora sus propiedades espumantes y emulsionantes. El presente proyecto propone investigar la capacidad de la L-glutaminasa de Bizionia argentinensis (una bacteria marina aislada de la península Antártica) para potenciar el sabor de hidrolizados proteicos. Para ello, se procedió al clonado, expresión recombinante, purificación y caracterización de esta enzima en condiciones variables de temperatura, pH, salinidad y solventes orgánicos. Los resultados mostraron que la enzima retuvo el 90% de la actividad máxima a 25ºC y a 3 M NaCl. Además, pudo incrementar el contenido de L-glutamato en hidrolizados proteicos. ARK/CAICYT: http://id.caicyt.gov.ar/ark:/s22504559/h8bbx2yq

Structural-Functional Characterization and Physiological Significance of Ferredoxin-NADP+ Reductase from Xanthomonas axonopodis pv. citri

Xanthomonas axonopodis pv. citri is a phytopathogen bacterium that causes severe citrus canker disease. Similar to other phytopathogens, after infection by this bacterium, plants trigger a defense mechanism that produces reactive oxygen species. Ferredoxin-NADP+ reductases (FNRs) are redox flavoenzymes that participate in several metabolic functions, including the response to reactive oxygen species. Xanthomonas axonopodis pv. citri has a gene (fpr) that encodes for a FNR (Xac-FNR) that belongs to the subclass I bacterial FNRs. The aim of this work was to search for the physiological role of this enzyme and to characterize its structural and functional properties. The functionality of Xac-FNR was tested by cross-complementation of a FNR knockout Escherichia coli strain, which exhibit high susceptibility to agents that produce an abnormal accumulation of •O2-. Xac-FNR was able to substitute for the FNR in E. coli in its antioxidant role. The expression of fpr in X. axonopodis pv. citri was assessed using semiquantitative RT-PCR and Western blot analysis. A 2.2-fold induction was observed in the presence of the superoxide-generating agents methyl viologen and 2,3-dimethoxy-1,4-naphthoquinone. Structural and functional studies showed that Xac-FNR displayed different functional features from other subclass I bacterial FNRs. Our analyses suggest that these differences may be due to the unusual carboxy-terminal region. We propose a further classification of subclass I bacterial FNRs, which is useful to determine the nature of their ferredoxin redox partners. Using sequence analysis, we identified a ferredoxin (XAC1762) as a potential substrate of Xac-FNR. The purified ferredoxin protein displayed the typical broad UV-visible spectrum of [4Fe-4S] clusters and was able to function as substrate of Xac-FNR in the cytochrome c reductase activity. Our results suggest that Xac-FNR is involved in the oxidative stress response of Xanthomonas axonopodis pv. citri and performs its biological function most likely through the interaction with ferredoxin XAC1762

Swapping FAD binding motifs between plastidic and bacterial ferredoxin-NADP(H) reductases.

International audiencePlant-type ferredoxin-NADP(H) reductases (FNRs) are grouped in two classes, plastidic with an extended FAD conformation and high catalytic rates and bacterial with a folded flavin nucleotide and low turnover rates. The 112-123 β-hairpin from a plastidic FNR and the carboxy-terminal tryptophan of a bacterial FNR, suggested to be responsible for the FAD differential conformation, were mutually exchanged. The plastidic FNR lacking the β-hairpin was unable to fold properly. An extra tryptophan at the carboxy terminus, emulating the bacterial FNR, resulted in an enzyme with decreased affinity for FAD and reduced diaphorase and ferredoxin-dependent cytochrome c reductase activities. The insertion of the β-hairpin into the corresponding position of the bacterial FNR increased FAD affinity but did not affect its catalytic properties. The same insertion with simultaneous deletion of the carboxy-terminal tryptophan produced a bacterial chimera emulating the plastidic architecture with an increased k(cat) and an increased catalytic efficiency for the diaphorase activity and a decrease in the enzyme's ability to react with its substrates ferredoxin and flavodoxin. Crystallographic structures of the chimeras showed no significant changes in their overall structure, although alterations in the FAD conformations were observed. Plastidic and bacterial FNRs thus reveal differential effects of key structural elements. While the 112-123 β-hairpin modulates the catalytic efficiency of plastidic FNR, it seems not to affect the bacterial FNR behavior, which instead can be improved by the loss of the C-terminal tryptophan. This report highlights the role of the FAD moiety conformation and the structural determinants involved in stabilizing it, ultimately modulating the functional output of FNRs

Structural backgrounds for the formation of a catalytically competent complex with NADP(H) during hydride transfer in ferredoxin–NADP+ reductases

AbstractThe role of the highly conserved C266 and L268 of pea ferredoxin–NADP+ reductase (FNR) in formation of the catalytically competent complex of the enzyme with NADP(H) was investigated. Previous studies suggest that the volume of these side-chains, situated facing the side of the C-terminal Y308 catalytic residue not stacking the flavin isoalloxazine ring, may be directly involved in the fine-tuning of the catalytic efficiency of the enzyme. Wild-type pea FNR as well as single and double mutants of C266 and L268 residues were analysed by fast transient-kinetic techniques and their midpoint reduction potentials were determined. For the C266A, C266M and C266A/L268A mutants a significant reduction in the overall hydride transfer (HT) rates was observed along with the absence of charge-transfer complex formation. The HT rate constants for NADPH oxidation were lower than those for NADP+ reduction, reaching a 30-fold decrease in the double mutant. In agreement, these variants exhibited more negative midpoint potentials with respect to the wild-type enzyme. The three-dimensional structures of C266M and L268V variants were solved. The C266M mutant shows a displacement of E306 away from the relevant residue S90 to accommodate the bulky methionine introduced. The overall findings indicate that in FNR the volume of the residue at position 266 is essential to attain the catalytic architecture between the nicotinamide and isoalloxazine rings at the active site and, therefore, for an efficient HT process. In addition, flexibility of the 268–270 loop appears to be critical for FNR to achieve catalytically competent complexes with NADP(H)

Effectiveness of Trichoderma strains isolated from the rhizosphere of citrus tree to control Alternaria alternata, Colletotrichum gloeosporioides and Penicillium digitatum A21 resistant to pyrimethanil in post‐harvest oranges [Citrus sinensis L. (Osbeck)]

Aims: Penicillium digitatum, Alternaria alternata and Colletotrichum gloeosporiodies are pathogens responsible for large decays and production losses of citrus. They are commonly controlled by fungicides, whose excessive applications have led to the emergence of resistant P. digitatum strains. Alternative approaches are imperative for sustainable and environmental harmless citrus production, being biological control a promising strategy. The objective was to evaluate the potential of Trichoderma strains native from the rhizosphere of citrus trees to control these pathogens. Methods and Results: Seven strains were isolated and identified as Trichoderma harzianum, T. ghizhouense, T. atroviride and T. koningiopsis through morphological and molecular analysis. Five of them showed effective antagonist performance in vitro against the pathogens. The strain T. harzianum IC‐30 was the best biological control agent in vivo, obtaining a reduction of rot percentage around 80 % after three weeks of infection of oranges with P. digitatum A21 (resistant to pyrimethanil). This strain also showed the highest chitinase and glucanase activity. Conclusions: T. harzianum IC‐30 is an optimal antagonist for the control of green mold spreading and other pathogens in post‐harvest citrus fruits. Significance and impact: The strain combined with supplementary practices could lead to sustainable management of citrus fungal diseases, dispensing with synthetic fungicides.EEA ConcordiaFil: Ferreira, Flavia V. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Entre Ríos; ArgentinaFil: Herrmann Andrade, Andreina M. Universidad Nacional de Entre Ríos. Facultad de Ciencias de la Alimentación; ArgentinaFil: Calabrese, Carla D. Universidad Nacional de Entre Ríos. Facultad de Ciencias de la Alimentación; ArgentinaFil: Bello, Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Concordia; ArgentinaFil: Vazquez, Daniel Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Concordia; ArgentinaFil: Musumeci, Matías A. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y TranUniversidad Nacional de Entre Ríos. Facultad de Ciencias de la Alimentación; Argentinasferencia de Entre Ríos; Argentina

Mycofumigation of postharvest blueberries with volatile compounds from Trichoderma atroviride IC-11 is a promising tool to control rots caused by Botrytis cinerea

Botrytis cinerea, the causal agent of the gray mold, is a filamentous fungus that infects blueberries and can cause important production losses in postharvest storage. Considering that the use of synthetic fungicides is not allowed on blueberries in postharvest conditions, alternative and natural strategies are needed to control gray mold. The objective of this work was to evaluate the capability of volatile organic compounds (VOCs) produced by Trichoderma atroviride IC-11 to control B. cinerea growth in blueberries after harvest.These VOCs inhibited almost completely B. cinerea growth in vitro. The most abundant volatile compound was 6-pentyl-α-pyrone (6PP). In vitro assays with pure 6PP confirmed its antifungal activity. The incidence of gray mold was evaluated in blueberries inoculated with B. cinerea and exposed to volatiles of T. atroviride IC-11. Gray mold incidence among those stored in air at 20 °C for 14 days was 100%, while the incidence among the volatile-treated fruit was 17%. Gray mold incidence among those stored in air at 4 °C for 31 days was 82%, while the incidence among the volatile-treated fruit was 11%. T. atroviride IC-11 VOCs inhibited mycelial growth and conidia germination of B. cinerea. The binding of VOCs to the surface of hyphae caused their vacuolation and deterioration. Selective cytotoxicity of 6PP on B. cinerea was observed but not on human intestinal cells at specific concentrations that controlled gray mold. The postharvest mycofumigation of blueberries with T. atroviride IC-11 VOCs is a promising approach to protect these fruits from gray mold.EEA ConcordiaFil: Bello, Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Concordia; ArgentinaFil: Montironi, Ivana Dalila. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria. Cátedra de Farmacología; ArgentinaFil: Medina, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología de los Alimentos de Entre Ríos; ArgentinaFil: Medina, María Belén. Universidad Nacional de Entre Ríos. Facultad de Ciencias de la Alimentación; ArgentinaFil: Munitz, Martín Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología de los Alimentos de Entre Ríos; ArgentinaFil: Munitz, Martín Sebastián. Universidad Nacional de Entre Ríos. Facultad de Ciencias de la Alimentación; ArgentinaFil: Ferreira, Flavia Vanina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología de los Alimentos de Entre Ríos; ArgentinaFil: Williman, Celia. Universidad Nacional de Entre Ríos. Facultad de Ciencias de la Alimentación; ArgentinaFil: Vazquez, Daniel Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Concordia; ArgentinaFil: Cariddi, Noelia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Microbiología e Inmunología. Laboratorio de Inmunología; Argentina.Fil: Cariddi, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biotecnología Ambiental y Salud; ArgentinaFil: Cariddi, Noelia. Universidad Nacional de Río Cuarto. Instituto de Biotecnología Ambiental y Salud; ArgentinaFil: Musumeci, Matías Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología de los Alimentos de Entre Ríos; ArgentinaFil: Musumeci, Matías Alejandro. Universidad Nacional de Entre Ríos. Facultad de Ciencias de la Alimentación; Argentin