Alessandro Aliverti

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 ~30°, with the C4 atom 3 Å from the flavin N5 atom.

Structural and functional diversity of ferredoxin-NADP(+) reductases

Although all ferredoxin-NADP(+) reductases (FNRs) catalyze the same reaction, i.e. the transfer of reducing equivalents between NADP(H) and ferredoxin, they belong to two unrelated families of proteins: the plant-type and the glutathione reductase-type of FNRs. Aim of this review is to provide a general classification scheme for these enzymes, to be used as a framework for the comparison of their properties. Furthermore, we report on some recent findings, which significantly increased the understanding of the structure-function relationships of FNRs, i.e. the ability of adrenodoxin reductase and its homologs to catalyze the oxidation of NADP(+) to its 4-oxo derivative, and the properties of plant-type FNRs from non-photosynthetic organisms. Plant-type FNRs from bacteria and Apicomplexan parasites provide examples of novel ways of FAD- and NADP(H)-binding. The recent characterization of an FNR from Plasmodium falciparum brings these enzymes into the field of drug design.

Reconstitution of an apicoplast-localised electron transfer pathway involved in the isoprenoid biosynthesis of Plasmodium falciparum.

In the malaria parasite Plasmodium falciparum isoprenoid precursors are synthesised inside a plastid‐like organelle (apicoplast) by the mevalonate independent 1‐deoxy‐d‐xylulose‐5‐phosphate (DOXP) pathway. The last reaction step of the DOXP pathway is catalysed by the LytB enzyme which contains a [4Fe–4S] cluster. In this study, LytB of P. falciparum was shown to be catalytically active in the presence of an NADPH dependent electron transfer system comprising ferredoxin and ferredoxin‐NADP+ reductase. LytB and ferredoxin were found to form a stable protein complex. These data suggest that the ferredoxin/ferredoxin‐NADP+ reductase redox system serves as the physiological electron donor for LytB in the apicoplast of P. falciparum.

Structure of the mutant E92K of [2Fe-2S] ferredoxin I from Spinacia oleracea at 1.7 A resolution.

Ferredoxin I (Fd I) from Spinacia oleracea is composed of 97 amino-acid residues and a [2Fe-2S] cluster. The crystal structure of the E92K mutant of Fd I was solved by molecular replacement and refined to an R factor of 19.6% for 11755 reflections at 1.7 A resolution. The overall structure and the active centre of spinach Fd is highly conserved with respect to ferredoxins of known structure. The E92K mutation appears to disturb a hydrogen-bond network which stabilizes the loop bearing the [2Fe-2S] cluster. This observation provides a rationale for the reduced electron-transfer efficiency displayed by the E92K mutant. Inspection of the crystal packing reveals that the side chain of Lys92 is engaged in an intermolecular interaction with Asp26 of a symmetry-related molecule. This feature may explain why only the mutant E92K and not wild-type Fd I could be successfully crystallized.

Involvement of lysine‐88 of spinach ferredoxin‐NADP+ reductase in the interaction with ferredoxin

A mutant of spinach ferredoxin‐NADP+ reductase, in which Lys‐88 has been changed to glutamine, has been obtained by site‐directed mutagenesis. The mutant enzyme was fully active as a diaphorase, but partially impaired in ferredoxin‐dependent cytochrome c reductase activity. By steady‐state kinetics, the K m for ferredoxin of the K88Q enzyme was found to have increased 10‐fold, whereas the k cat was unaffected by the amino acid replacement. The interaction between oxidized ferredoxin and the enzyme forms was also studied by spectrofluorimetric titration:K d values of 110 and 10 nM were determined for the mutant and wild‐type proteins, respectively. These data point out the importance of a positive charge at position 88 of the reductase for the interaction with ferredoxin, confirming previous cross‐linking studies.

The Plant-Type Ferredoxin-NADP + Reductase/Ferredoxin Redox System as a Possible Drug Target Against Apicomplexan Human Parasites

Apicomplexa are unicellular, obligate intracellular parasites of great medical importance. They include human pathogens like Plasmodium spp., the causative agent of malaria, and Toxoplasma gondii, an opportunistic parasite of immunosuppressed individuals and a common cause of congenital disease (toxoplasmosis). They alone affect several hundred million people worldwide so that new drugs, especially for plasmodial infections, are urgently needed. This review will focus on a recently emerged, potential drug target, a plant-type redox system consisting of ferredoxin-NADP(+) reductase (FNR) and its redox partner, ferredoxin (Fd). Both reside in an unique organelle of these parasites, named apicoplast, which is of algal origin. The apicoplast has been shown to be required for pathogen survival. In addition to other pathways already identified in this compartment, the FNR/Fd redox system represents a promising drug target because homologous proteins are not present in host organisms. Furthermore, a wealth of structural information exists on the closely related plant proteins, which can be exploited for structure-function studies of the apicomplexan protein pair. T. gondii and P. falciparum FNRs have been cloned, and the T. gondii enzyme was shown to be a flavoprotein active as a NADPH-dependent oxidoreductase. Both phylogenetic and biochemical analyses indicate that T. gondii FNR is similar in function to the isoform present in non-photosynthetic plastids whereby electron flow is from NADPH to oxidized Fd. The resulting reduced Fd is then presumably used as a reductant for various target enzymes whose nature is just starting to emerge. Among the likely candidates is the iron-sulfur cluster biosynthesis pathway, which is also located in the apicoplast and dependent on reducing power. Furthermore, lipoic acid synthase and enzymes of the isoprenoid biosynthetic pathway may be other conceivable targets. Since all these metabolic steps are vital for the parasite, blocking electron flow from FNR to Fd by inhibition of either FNR activity or its molecular interaction with Fd should also interfere with these pathways, ultimately killing the parasite. Although the three-dimensional structure of FNR from T. gondii is not yet known, experimental and computational evidence shows that apicomplexan and plant enzymes are very similar in structure. Furthermore, single amino acid changes can have profound effects on the enzyme activity and affinity for Fd. This knowledge may be exploited for the design of inhibitors of protein-protein interaction. On the other hand, specifically tailored NAD(P) analogues or mimetics based on previously described substances might be useful lead compounds for apicomplexan FNR inhibitors.

Ferredoxin-NADP+ reductase and ferredoxin of the protozoan parasite Toxoplasma gondii interact productively in Vitro and in Vivo.

Toxoplasma gondii possesses an apicoplast-localized, plant-type ferredoxin-NADP+reductase. We have cloned a [2Fe-2S] ferredoxin from the same parasite to investigate the interplay of the two redox proteins. A detailed characterization of the two purified recombinant proteins, particularly as to their interaction, has been performed. The two-protein complex was able to catalyze electron transfer from NADPH to cytochrome c with high catalytic efficiency. The redox potential of the flavin cofactor (FAD/FADH−) of the reductase was shown to be more positive than that of the NADP+/NADPH couple, thus favoring electron transfer from NADPH to yield reduced ferredoxin. The complex formation between the reductase and ferredoxins from various sources was studied bothin vitro by several approaches (enzymatic activity, cross-linking, protein fluorescence quenching, affinity chromatography) and in vivo by the yeast two-hybrid system. Our data show that the two proteins yield an active complex with high affinity, strongly suggesting that the two proteins of T. gondiiform a physiological redox couple that transfers electrons from NADPH to ferredoxin, which in turn is used by some reductive biosynthetic pathway(s) of the apicoplast. These data provide the basis for the exploration of this redox couple as a drug target in apicomplexan parasites.

Synthesis of human renalase1 in Escherichia coli and its purification as a FAD-containing holoprotein

Renalase is a protein ubiquitous in vertebrates, which has been proposed to modulate blood pressure and heart rate, and whose downregulation might result in hypertension. Despite its potential relevance for human health, the biochemical characterization of renalase is still lacking, possibly due to difficulties in obtaining it in recombinant form. By expressing two different gene constructs, we found that the major isoform of human renalase, renalase1, is mainly produced in Escherichia coli in inclusion bodies. However, by optimizing the expression conditions, significant amounts of soluble products were obtained. Both soluble renalase forms have been purified to homogeneity exploiting their N-terminal His-tag. Linking of the protein of interest to the SUMO protein did not improve solubility, but yielded untagged renalase1 after proteolytic processing of the fusion product. The two recombinant renalase forms displayed the same molecular properties. They bind equimolar amounts of FAD and appear to be correctly folded by various criteria. The procedures for the production and isolation of recombinant renalase1 here reported are expected to boost the much awaited biochemical studies on this remarkable protein.

Direct electrochemistry and EPR spectroscopy of spinach ferredoxin mutants with modified electron transfer properties.

Mutations of the conserved residue Glu‐92 to lysine, glutamine, and alanine have been performed in the recombinant ferredoxin I of spinach leaves. The purified ferredoxin mutants were found twice as active with respect to wild‐type protein in the NADPH‐cytochrome c reductase reaction catalyzed by ferredoxin‐NADP+ reductase in the presence of ferredoxin. Cyclic voltammetry and EPR measurements showed that the mutations cause a change in the [2Fe‐2S] cluster geometry, whose redox potential becomes approximately 80 mV less negative. These data point to a role of the Glu‐92 side‐chain in determining the low redox potential typical of the [2Fe‐2S] cluster of chloroplast and cyanobacterial ferredoxins. Also a ferredoxin/ferredoxin‐NADP+ reductase chimeric protein obtained by gene fusion was overproduced in Escherichia coli and purified. Fusion of the ferredoxin with its reductase causes only minor effects to the iron‐sulfur cluster, as judged by cyclic voltammetry and EPR measurements.

Guanidinium chloride- and urea-induced unfolding of FprA, a mycobacterium NADPH-ferredoxin reductase stabilization of an apo-protein by GdmCl

The guanidinium chloride‐ and urea‐induced unfolding of FprA, a mycobacterium NADPH‐ferredoxin reductase, was examined in detail using multiple spectroscopic techniques, enzyme activity measurements and size exclusion chromatography. The equilibrium unfolding of FprA by urea is a cooperative process where no stabilization of any partially folded intermediate of protein is observed. In comparison, the unfolding of FprA by guanidinium chloride proceeds through intermediates that are stabilized by interaction of protein with guanidinium chloride. In the presence of low concentrations of guanidinium chloride the protein undergoes compaction of the native conformation; this is due to optimization of charge in the native protein caused by electrostatic shielding by the guanidinium cation of charges on the polar groups located on the protein side chains. At a guanidinium chloride concentration of about 0.8 m, stabilization of apo‐protein was observed. The stabilization of apo‐FprA by guanidinium chloride is probably the result of direct binding of the Gdm+ cation to protein. The results presented here suggest that the difference between the urea‐ and guanidinium chloride‐induced unfolding of FprA could be due to electrostatic interactions stabilizating the native conformation of this protein.