Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs, EC 1.18.1.2) are a widely distributed class of flavoenzymes that have non-covalently bound FAD cofactor as a redox center. FNRs participate in a wide variety of redox-based metabolic reactions, transferring electrons between obligatory one-and two-electron carriers and therefore functioning as a general electron splitter. In non-phototrophic bacteria and eukaryotes, the reaction is driven towards ferredoxin (Fd) reduction, providing reducing power for multiple metabolic pathways, including steroid hydroxylation in mammalian mitochondria, nitrite reduction and glutamate synthesis in heterotrophic tissues of vascular plants, radical propagation and scavenging in prokaryotes, and hydrogen and nitrogen fixation in anaerobes (for a review, see [1,2]). In plants, FNR participates in photosynthetic electron transport, reducing Fd at the level of photosystem I, and transferring electrons to NADP + . This process ends with the formation of the NADPH necessary for CO 2 fixation and other biosynthetic pathways [2].The three-dimensional structures of several FNRs have been determined. They display similar structural features, which have been defined as the prototype for a large family of flavoenzymes [3][4][5][6][7][8][9][10] Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliver NADPH or low-potential one-electron donors (ferredoxin, flavodoxin, adrenodoxin) to redox-based metabolic reactions in plastids, mitochondria and bacteria. Plastidic FNRs are quite efficient reductases. In contrast, FNRs from organisms possessing a heterotrophic metabolism or anoxygenic photosynthesis display turnover numbers 20-to 100-fold lower than those of their plastidic and cyanobacterial counterparts. Several structural features of these enzymes have yet to be explained. The residue Y308 in pea FNR is stacked nearly parallel to the re-face of the flavin and is highly conserved amongst members of the family. By computing the relative free energy for the lumiflavin-phenol pair at different angles with the relative position found for Y308 in pea FNR, it can be concluded that this amino acid is constrained against the isoalloxazine. This effect is probably caused by amino acids C266 and L268, which face the other side of this tyrosine. Simple and double FNR mutants of these amino acids were obtained and characterized. It was observed that a decrease or increase in the amino acid volume resulted in a decrease in the catalytic efficiency of the enzyme without altering the protein structure. Our results provide experimental evidence that the volume of these amino acids participates in the fine-tuning of the catalytic efficiency of the enzyme.
Background: Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron transfer between NADP(H) and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR) displays low sequence identity with plant (34% with Zea mays) and bacterial (31% with Escherichia coli) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs.
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.
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