A Highly Stable Plastidic-Type Ferredoxin-NADP(H) Reductase in the Pathogenic Bacterium Leptospira interrogans Daniela L. Catalano-Dupuy, Matı´as A. Musumeci, Arleth Lo ´ pez-Rivero, Eduardo A. Ceccarelli* Molecular Biology Division, Instituto de Biologı ´a Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquı ´micas y Farmace ´ uticas, Universidad Nacional de Rosario, Rosario, Argentina 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. Citation: Catalano-Dupuy DL, Musumeci MA, Lo ´ pez-Rivero A, Ceccarelli EA (2011) A Highly Stable Plastidic-Type Ferredoxin-NADP(H) Reductase in the Pathogenic Bacterium Leptospira interrogans. PLoS ONE 6(10): e26736. doi:10.1371/journal.pone.0026736 Editor: Anil Kumar Tyagi, University of Delhi, India Received April 26, 2011; Accepted October 3, 2011; Published October 24, 2011 Copyright: ß 2011 Catalano-Dupuy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants PICT-2007 01-00645 and 00739, ANCYPT, Ministerio de Ciencia, Tecnologı ´a e Innovacio ´ n Productiva, Argentina (www.agencia.mincyt.gov.ar); PIP 252 and 114-200901-00337 from CONICET Consejo Nacional de Investigaciones Cientı ´ficas y Te ´cnicas, Argentina (www.conicet. gov.ar); FBBEI2/08 from Fundacio ´ n Bunge y Born, Argentina (http://www.fundacionbyb.org); and BIO187 from the University of Rosario, Argentina (www.unr.edu. ar.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ceccarelli@ibr.gov.ar Introduction Ferredoxin-NADP(H) reductases (FNRs) are ubiquitous mono- meric enzymes that contain a noncovalently bound FAD as a prosthetic group. To date, the participation of FNRs has been documented in processes as dissimilar as photosynthesis, steroid hydroxylation, nitrate reduction, anaerobic pyruvate assimilation and fatty acid desaturation. The involvement of FNR has been also documented in xenobiotic detoxification, amino acid and deoxyribonucleotide synthesis, iron-sulfur cluster biogenesis and in the regulation of several metabolic pathways [1–4]. The biological catalytic FNR activity comprises the reversible electron transfer between NADP(H) and different low potential one-electron donors (e.g., ferredoxin, flavodoxin, adrenodoxin, heme-oxygenase and iron) [1–7]. The aforementioned reductase activities are performed, in nature, by enzymes that belong to two unrelated protein families [2,3]. One of these families includes adrenodoxin reductases, some bacterial homologues and bacterial oxygenase-coupled NADH- ferredoxin reductases. Enzymes of the above group have been found mainly in the mitochondria of eukaryotic organisms and in some bacteria such as Mycobacterium tuberculosis and Pseudomonas. The other group, known as the plant-type FNR family, contains FNRs that are easily identified by the presence of highly conserved amino acid clusters located in the FAD and NADP(H) binding domains [2]. This group is subdivided into bacterial and plastidic FNR classes. Whereas plastidic FNRs display turnover numbers that are related to the photosynthesis needs (200–600 s 21 ), bacterial FNRs are much less active, with turnover numbers that are 20- to 100-fold lower than those of their plastidic counterparts [1,2,8]. The low sequence homology between both subgroups (plastidic and bacterial FNRs) weakens the possibility of performing phylogenetic analyses of all group members; however, a common ancestor for all plant-type FNRs can be expected. We have previously assigned the FNR from Leptospira interrogans (LepFNR) to the plastidic FNR class [2], and this was confirmed by resolution of the LepFNR crystal structure [9]. The presence of a plastidic FNR in Leptospira might be explained as the result of lateral gene transfer, and this was probably selected to provide the bacterium PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26736