Crystal structure of the FAD-containing ferredoxin-NADP+ reductase from the plant pathogen Xanthomonas axonopodis pv. citri

We have solved the structure of ferredoxin-NADP(H) reductase, FPR, from the plant pathogen Xanthomonas axonopodis pv. citri, responsible for citrus canker, at a resolution of 1.5¿Å. This structure reveals differences in the mobility of specific loops when compared to other FPRs, probably unrelated to the hydride transfer process, which contributes to explaining the structural and functional divergence between the subclass I FPRs. Interactions of the C-terminus of the enzyme with the phosphoadenosine of the cofactor FAD limit its mobility, thus affecting the entrance of nicotinamide into the active site. This structure opens the possibility of rationally designing drugs against the X. axonopodis pv. citri phytopathogen

Identification of inhibitors targeting ferredoxin-NADP+ reductase from the xanthomonas citri subsp. Citri phytopathogenic bacteria

Ferredoxin-NADP(H) reductases (FNRs) deliver NADPH or low potential one-electron donors to redox-based metabolism in plastids and bacteria. Xanthomonas citri subsp. citri (Xcc) is a Gram-negative bacterium responsible for citrus canker disease that affects commercial citrus crops worldwide. The Xcc fpr gene encodes a bacterial type FNR (XccFPR) that contributes to the bacterial response to oxidative stress conditions, usually found during plant colonization. Therefore, XccFPR is relevant for the pathogen survival and its inhibition might represent a strategy to treat citrus canker. Because of mechanistic and structural differences from plastidic FNRs, XccFPR is also a potential antibacterial target. We have optimized an activity-based high-throughput screening (HTS) assay that identifies XccFPR inhibitors. We selected 43 hits from a chemical library and narrowed them down to the four most promising inhibitors. The antimicrobial effect of these compounds was evaluated on Xcc cultures, finding one with antimicrobial properties. Based on the functional groups of this compound and their geometric arrangement, we identified another three XccFPR inhibitors. Inhibition mechanisms and constants were determined for these four XccFPR inhibitors. Their specificity was also evaluated by studying their effect on the plastidic Anabaena PCC 7119 FNR, finding differences that can become interesting tools to discover Xcc antimicrobials

Dynamics of the active site architecture in plant-type ferredoxin-NADP + reductases catalytic complexes

Kinetic isotope effects in reactions involving hydride transfer and their temperature dependence are powerful tools to explore dynamics of enzyme catalytic sites. In plant-type ferredoxin-NADP+ reductases the FAD cofactor exchanges a hydride with the NADP(H) coenzyme. Rates for these processes are considerably faster for the plastidic members (FNR) of the family than for those belonging to the bacterial class (FPR). Hydride transfer (HT) and deuteride transfer (DT) rates for the NADP+ coenzyme reduction of four plant-type FNRs (two representatives of the plastidic type FNRs and the other two from the bacterial class), and their temperature dependences are here examined applying a full tunnelling model with coupled environmental fluctuations. Parameters for the two plastidic FNRs confirm a tunnelling reaction with active dynamics contributions, but isotope effects on Arrhenius factors indicate a larger contribution for donor-acceptor distance (DAD) dynamics in the Pisum sativum FNR reaction than in the Anabaena FNR reaction. On the other hand, parameters for bacterial FPRs are consistent with passive environmental reorganisation movements dominating the HT coordinate and no contribution of DAD sampling or gating fluctuations. This indicates that active sites of FPRs are more organised and rigid than those of FNRs. These differences must be due to adaptation of the active sites and catalytic mechanisms to fulfil their particular metabolic roles, establishing a compromise between protein flexibility and functional optimisation. Analysis of site-directed mutants in plastidic enzymes additionally indicates the requirement of a minimal optimal architecture in the catalytic complex to provide a favourable gating contribution. © 2014 Elsevier B.V.Peer Reviewe

A productive NADP+ binding mode of ferredoxin-NADP+ reductase revealed by protein engineering and crystallographic studies

The flavoenzyme ferredoxin-NADP+ reductase (FNR) catalyzes the production of NADPH during photosynthesis. Whereas the structures of FNRs from spinach leaf and a cyanobacterium as well as many of their homologs have been solved, none of these studies has yielded a productive geometry of the flavin-nicotinamide interaction. Here, we show that this failure occurs because nicotinamide binding to wild type FNR involves the energetically unfavorable displacement of the C-terminal Tyr side chain. We used mutants of this residue (Tyr 308) of pea FNR to obtain the structures of productive NADP+ and NADPH complexes. These structures reveal a unique NADP+ binding mode in which the nicotinamide ring is not parallel to the flavin isoalloxazine ring, but lies against it at an angle of approximately 30 degrees, with the C4 atom 3 A from the flavin N5 atom

Interspecific Potato Breeding Lines Display Differential Colonization Patterns and Induced Defense Responses after Ralstonia solanacearum Infection

Potato (Solanum tuberosum L.) is one of the main hosts of Ralstonia solanacearum, the causative agent of bacterial wilt. This plant pathogen bacteria produce asymptomatic latent infections that promote its global spread, hindering disease control. A potato breeding program is conducted in Uruguay based on the introgression of resistance from the wild native species S. commersonii Dun. Currently, several backcrosses were generated exploiting the high genetic variability of this wild species resulting in advanced interspecific breeding lines with different levels of bacterial wilt resistance. The overall aim of this work was to characterize the interaction of the improved potato germplasm with R. solanacearum. Potato clones with different responses to R. solanacearum were selected, and colonization, dissemination and multiplication patterns after infection were evaluated. A R. solanacearum strain belonging to the phylotype IIB-sequevar 1, with high aggressiveness on potato was genetically modified to constitutively generate fluorescence and luminescence from either the green fluorescence protein gene or lux operon. These reporter strains were used to allow a direct and precise visualization of fluorescent and luminescent cells in plant tissues by confocal microscopy and luminometry. Based on wilting scoring and detection of latent infections, the selected clones were classified as susceptible or tolerant, while no immune-like resistance response was identified. Typical wilting symptoms in susceptible plants were correlated with high concentrations of bacteria in roots and along the stems. Tolerant clones showed a colonization pattern restricted to roots and a limited number of xylem vessels only in the stem base. Results indicate that resistance in potato is achieved through restriction of bacterial invasion and multiplication inside plant tissues, particularly in stems. Tolerant plants were also characterized by induction of anatomical and biochemical changes after R. solanacearum infection, including hyperplasic activity of conductor tissue, tylose production, callose and lignin deposition, and accumulation of reactive oxygen species. This study highlights the potential of the identified tolerant interspecific potato clones as valuable genetic resources for potato-breeding programs and leads to a better understanding of resistance against R. solanacearum in potato