Ferredoxin-NADP+ Reductase from Plasmodium falciparum Undergoes NADP+-dependent Dimerization and Inactivation: Functional and Crystallographic Analysis

Milani, Mario; Balconi, E.; Aliverti, Alessandro; Mastrangelo, Eloise; Seeber, Frank; Bolognesi, Martino; Zanetti, G.

doi:10.1016/j.jmb.2007.01.005

Cited by 40 publications

(84 citation statements)

References 43 publications

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“…Spectrophotometric active-site titrations of renalase with NADP + , NAD + , 2′-P-AMP, or 5′-AMP were carried out at 25°C in 50 mM Tris-HCl (pH 7.4), according to the procedure described elsewhere. 31 NADH-dependent and NADPHdependent diaphorase activities of renalase were assayed at 37°C in 50 mM Tris-HCl (pH 7.2), using a fixed concentration of either 100 μM INT or 500 μM WST1 as electron acceptor. The reaction mixture included 2.6 μM renalase and a variable concentration of reduced dinucleotide in the range of 0.05-2 mM.…”

Section: Renalase Expression and Purificationmentioning

confidence: 99%

FAD-Binding Site and NADP Reactivity in Human Renalase: A New Enzyme Involved in Blood Pressure Regulation

Milani

Ciriello

Baroni

et al. 2011

Journal of Molecular Biology

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Renalase is a recently discovered flavoprotein that regulates blood pressure, regulates sodium and phosphate excretion, and displays cardioprotectant action through a mechanism that is barely understood to date. It has been proposed to act as a catecholamine-degrading enzyme, via either O(2)-dependent or NADH-dependent mechanisms. Here we report the renalase crystal structure at 2.5 Å resolution together with new data on its interaction with nicotinamide dinucleotides. Renalase adopts the p-hydroxybenzoate hydroxylase fold topology, comprising a Rossmann-fold-based flavin adenine dinucleotide (FAD)-binding domain and a putative substrate-binding domain, the latter of which contains a five-stranded anti-parallel β-sheet. A large cavity (228 Å(3)), facing the flavin ring, presumably represents the active site. Compared to monoamine oxidase or polyamine oxidase, the renalase active site is fully solvent exposed and lacks an 'aromatic cage' for binding the substrate amino group. Renalase has an extremely low diaphorase activity, displaying lower k(cat) but higher k(cat)/K(m) for NADH compared to NADPH. Moreover, its FAD prosthetic group becomes slowly reduced when it is incubated with NADPH under anaerobiosis, and binds NAD(+) or NADP(+) with K(d) values of ca 2 mM. The absence of a recognizable NADP-binding site in the protein structure and its poor affinity for, and poor reactivity towards, NADH and NADPH suggest that these are not physiological ligands of renalase. Although our study does not answer the question on the catalytic activity of renalase, it provides a firm framework for testing hypotheses on the molecular mechanism of its action.

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Section: Renalase Expression and Purificationmentioning

confidence: 99%

FAD-Binding Site and NADP Reactivity in Human Renalase: A New Enzyme Involved in Blood Pressure Regulation

Milani

Ciriello

Baroni

et al. 2011

Journal of Molecular Biology

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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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“…Interestingly, the lysine residues in spinach FNR are located mainly on the water exposed surface of the molecule (Bruns and Karplus 1995), and they could promote conformational changes, as has been observed for pH dependent changes in synthetic poly-L lysine (Greenfield and Fasman 1969). Milani et al (2007) showed that FNR from Plasmodium falciparum creates stable, inactive aggregates via a disulphide bond formed by Cys 99 from two interacting peptides. The cysteine at that (or nearest) position is absent in both wheat FNRs and the disulphide bond may not stabilize the observed oligomer.…”

Section: Properties Of Two Fnr Isoforms From Wheatmentioning

confidence: 80%

“…The cysteine at that (or nearest) position is absent in both wheat FNRs and the disulphide bond may not stabilize the observed oligomer. However, Milani et al (2007) showed also the quaternary assembly they observed in crystals, which was formed by some additional interaction between the a-helices of the C-terminal domains. The oligomerization of wheat FNRs might result in an observed increase of b-structure, due to the formation of b-like hydrogen binding between helices.…”

Section: Properties Of Two Fnr Isoforms From Wheatmentioning

confidence: 90%

Two isoforms of ferredoxin:NADP+ oxidoreductase from wheat leaves: purification and initial biochemical characterization

et al. 2008

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Ferredoxin:NADP(+) oxidoreductase is an enzyme associated with the stromal side of the thylakoid membrane in the chloroplast. It is involved in photosynthetic linear electron transport to produce NADPH and is supposed to play a role in cyclic electron transfer, generating a transmembrane pH gradient allowing ATP production, if photosystem II is non-functional or no NADP(+) is available for reduction. Different FNR isoforms have been described in non-photosynthetic tissues, where the enzyme catalyses the NADPH-dependent reduction of ferredoxin (Fd), necessary for some biosynthetic pathways. Here, we report the isolation and purification of two FNR isoproteins from wheat leaves, called FNR-A and FNR-B. These forms of the enzyme were identified as products of two different genes, as confirmed by mass spectrometry. The molecular masses of FNR-A and FNR-B were 34.3 kDa and 35.5 kDa, respectively. The isoelectric point of both FNR-A and FNR-B was about 5, but FNR-B appeared more acidic (of about 0.2 pH unit) than FNR-A. Both isoenzymes were able to catalyse a NADPH-dependent reduction of dibromothymoquinone and the mixture of isoforms catalysed reduction of cytochrome c in the presence of Fd. For the first time, the pH- and ionic strength dependent oligomerization of FNRs is observed. No other protein was necessary for complex formation. The putative role of the two FNR isoforms in photosynthesis is discussed based on current knowledge of electron transport in chloroplasts.

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Ferredoxin-NADP+ Reductase from Plasmodium falciparum Undergoes NADP+-dependent Dimerization and Inactivation: Functional and Crystallographic Analysis

Cited by 40 publications

References 43 publications

FAD-Binding Site and NADP Reactivity in Human Renalase: A New Enzyme Involved in Blood Pressure Regulation

FAD-Binding Site and NADP Reactivity in Human Renalase: A New Enzyme Involved in Blood Pressure Regulation

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

Two isoforms of ferredoxin:NADP+ oxidoreductase from wheat leaves: purification and initial biochemical characterization

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