Showing papers by "Eduardo A. Ceccarelli published in 2011"

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

Abstract: Plant-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 kcat and an incre...

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

Abstract: 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.

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

Abstract: 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 (•)O(2)(-) 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 22-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