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.
Highlights
Ferredoxin-NADP(H) reductases (FNRs) are ubiquitous monomeric enzymes that contain a noncovalently bound FAD as a prosthetic group
Sequence alignment and phylogeny of plastidic FNRs The complete DNA database that is available at the NCBI was searched using the tBLASTn method with the FNR amino acid sequences from L. interrogans, the mature region of the Pisum sativum from leaf, Escherichia coli and Rhodobacter capsulatus as query sequences
We identified a component of the aromatic degradation pathway that is found in the Betaproteobacterias of the Rhodocyclaceae family (i.e., BoxAB) of Azoarcus evansii [17] and in members of the order Burkholderiales (i.e., Bordetella, Burkholderia, which was previously a part of Pseudomonas, Ralstonia, Variovorax, etc.) (Fig. 1)
Summary
Ferredoxin-NADP(H) reductases (FNRs) are ubiquitous monomeric enzymes that contain a noncovalently bound FAD as a prosthetic group. To assess whether LepFNR interacts with protein substrates from other sources, cytochrome c reductase activity was measured using P. sativum leaf ferredoxin.
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