A single in vivo‐selected point mutation in the active center of <i>Toxoplasma gondii</i> ferredoxin‐NADP<sup>+</sup> reductase leads to an inactive enzyme with greatly enhanced affinity for ferredoxin

Thomsen‐Zieger, Nadine; Pandini, V.; Caprini, G.; Aliverti, Alessandro; Cramer, Jörg; Selzer, Paul M.; Zanetti, G.; Seeber, Frank

doi:10.1016/j.febslet.2004.09.042

Cited by 16 publications

(20 citation statements)

References 27 publications

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“…In PfFNR, the bound FAD possesses an E m suited for electron flow towards the [2Fe–2S] cluster of PfFd [14]. K m and K d values for PfFd are similar to those reported for other plastidic‐type FNR/Fd couples [10,29,30]. On the other hand, FAD semiquinone is highly destabilized in PfFNR.…”

Section: Discussionmentioning

confidence: 79%

The ferredoxin‐NADP⁺ reductase/ferredoxin electron transfer system of Plasmodium falciparum

et al. 2009

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In the apicoplast of apicomplexan parasites, plastidic-type ferredoxin and ferredoxin-NADP + reductase (FNR) form a short electron transport chain that provides reducing power for the synthesis of isoprenoid precursors. These proteins are attractive targets for the development of novel drugs against diseases such as malaria, toxoplasmosis, and coccidiosis. We have obtained ferredoxin and FNR of both Toxoplasma gondii and Plasmodium falciparum in recombinant form, and recently we solved the crystal structure of the P. falciparum reductase. Here we report on the functional properties of the latter enzyme, which differ markedly from those of homologous FNRs. In the physiological reaction, P. falciparum FNR displays a k cat five-fold lower than those usually determined for plastidic-type FNRs. By rapid kinetics, we found that hydride transfer between NADPH and proteinbound FAD is slower in the P. falciparum enzyme. The redox properties of the enzyme were determined, and showed that the FAD semiquinone species is highly destabilized. We propose that these two features, i.e. slow hydride transfer and unstable FAD semiquinone, are responsible for the poor catalytic efficiency of the P. falciparum enzyme. Another unprecedented feature of the malarial parasite FNR is its ability to yield, under oxidizing conditions, an inactive dimeric form stabilized by an intermolecular disulfide bond. Here we show that the monomer-dimer interconversion can be controlled by oxidizing and reducing agents that are possibly present within the apicoplast, such as H 2 O 2 , glutathione, and lipoate. This finding suggests that modulation of the quaternary structure of P. falciparum FNR might represent a regulatory mechanism, although this needs to be verified in vivo.

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Section: Discussionmentioning

confidence: 79%

The ferredoxin‐NADP⁺ reductase/ferredoxin electron transfer system of Plasmodium falciparum

et al. 2009

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“…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 with some adaptive advantages. Plastidic-type FNRs have been also found in Plasmodium falciparum [10], [11] and Toxoplasma gondii [12], [13].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2011

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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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“…This is the case, for example, in nonphotosynthetic tissues of plants (12,13) and in heterocysts of cyanobacteria (14), where isoforms have redox potentials that favor reverse electron flow. Fd and FNR are also found in the apicoplast of apicomplexan parasites (15) where they could also function in the reverse direction with Fd red possibly involved in the biosynthesis of iron-sulfur clusters (16). Whether it is reduced by PSI or by FNR/NADPH, Fd has been also reported to be involved in processes such as reduction of protons to hydrogen in some green algae (17), chlorophyll catabolism (18), glycine betaine synthesis (19), phytochrome biosynthesis (20), synthesis of phycobiliproteins in cyanobacteria (21), fatty acid desaturase (22), and nitrogen fixation in cyanobacterial heterocysts (23).…”

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confidence: 99%

Ferredoxin-NADP+ Reductase

Cassan

Lagoutte

Sétif

2005

Journal of Biological Chemistry

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The electron transfer cascade from photosystem I to NADP ؉ was studied at physiological pH by flash-absorption spectroscopy in a Synechocystis PCC6803 reconstituted system comprised of purified photosystem I, ferredoxin, and ferredoxin-NADP ؉ reductase. Experiments were conducted with a 34-kDa ferredoxin-NADP ؉ reductase homologous to the chloroplast enzyme and a 38-kDa N-terminal extended form. Small differences in kinetic and catalytic properties were found for these two forms, although the largest one has a 3-fold decreased affinity for ferredoxin. The dissociation rate of reduced ferredoxin from photosystem I (800 s ؊1 ) and the redox potential of the first reduction of ferredoxin-NADP ؉ reductase (؊380 mV) were determined. In the absence of NADP ؉ , differential absorption spectra support the existence of a high affinity complex between oxidized ferredoxin and semireduced ferredoxin-NADP ؉ reductase. An effective rate of 140 -170 s ؊1 was also measured for the second reduction of ferredoxin-NADP ؉ reductase, this process having a rate constant similar to that of the first reduction. In the presence of NADP ؉ , the second-order rate constant for the first reduction of ferredoxin-NADP ؉ reductase was 20% slower than in its absence, in line with the existence of ternary complexes (ferredoxin-NADP ؉ reductase)-NADP ؉ -ferredoxin. A single catalytic turnover was monitored, with 50% NADP ؉ being reduced in 8 -10 ms using 1.6 M photosystem I. In conditions of multiple turnover, we determined initial rates of 360 -410 electrons per s and per ferredoxin-NADP ؉ reductase for the reoxidation of 3.5 M photoreduced ferredoxin. Identical rates were found with photosystem I lacking the PsaE subunit and wild type photosystem I. This suggests that, in contrast with previous proposals, the PsaE subunit is not involved in NADP ؉ photoreduction.

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A single in vivo‐selected point mutation in the active center of Toxoplasma gondii ferredoxin‐NADP⁺ reductase leads to an inactive enzyme with greatly enhanced affinity for ferredoxin

Cited by 16 publications

References 27 publications

The ferredoxin‐NADP⁺ reductase/ferredoxin electron transfer system of Plasmodium falciparum

The ferredoxin‐NADP⁺ reductase/ferredoxin electron transfer system of Plasmodium falciparum

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

Ferredoxin-NADP+ Reductase

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A single in vivo‐selected point mutation in the active center of Toxoplasma gondii ferredoxin‐NADP+ reductase leads to an inactive enzyme with greatly enhanced affinity for ferredoxin

Cited by 16 publications

References 27 publications

The ferredoxin‐NADP+ reductase/ferredoxin electron transfer system of Plasmodium falciparum

The ferredoxin‐NADP+ reductase/ferredoxin electron transfer system of Plasmodium falciparum

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

Ferredoxin-NADP+ Reductase

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A single in vivo‐selected point mutation in the active center of Toxoplasma gondii ferredoxin‐NADP⁺ reductase leads to an inactive enzyme with greatly enhanced affinity for ferredoxin

The ferredoxin‐NADP⁺ reductase/ferredoxin electron transfer system of Plasmodium falciparum

The ferredoxin‐NADP⁺ reductase/ferredoxin electron transfer system of Plasmodium falciparum