Andrea Bellelli

Inhibition of Schistosoma mansoni thioredoxin-glutathione reductase by auranofin: structural and kinetic aspects.

Schistosomiasis is a parasitic disease affecting over 200 million people currently treated with one drug, praziquantel. A possible drug target is the seleno-protein thioredoxin-glutathione reductase (TGR), a key enzyme in the pathway of the parasite for detoxification of reactive oxygen species. The enzyme is a unique fusion of a glutaredoxin domain with a thioredoxin reductase domain, which contains a selenocysteine (Sec) as the penultimate amino acid. Auranofin (AF), a gold-containing compound already in clinical use as an anti-arthritic drug, has been shown to inhibit TGR and to substantially reduce worm burden in mice. Using x-ray crystallography we solved (at 2.5 Å resolution) the structure of wild type TGR incubated with AF. The electron density maps show that the actual inhibitor is gold, released from AF. Gold is bound at three different sites not directly involving the C-terminal Sec residue; however, because the C terminus in the electron density maps is disordered, we cannot exclude the possibility that gold may also bind to Sec. To investigate the possible role of Sec in the inactivation kinetics, we tested the effect of AF on a model enzyme of the same superfamily, i.e. the naturally Sec-lacking glutathione reductase, and on truncated TGR. We demonstrate that the role of selenium in the onset of inhibition by AF is catalytic and can be mimicked by an external source of selenium (benzeneselenol). Therefore, we propose that Sec mediates the transfer of gold from its ligands in AF to the redox-active Cys couples of TGR.

Modulation of mitochondrial respiration by nitric oxide: investigation by single cell fluorescence microscopy

With the electro‐driven import of rhodamine 123, we used single cell fluorescence microscopy to single out the contribution of nitric oxide (NO) in controlling mitochondrial membrane potential expressed by (stationary growing) rhabdomyosarcoma and neuroblastoma cells in culture. The experimental design and the computer‐aided image analysis detected and quantitated variations of fluorescence signals specific to mitochondria. We observed that 1) the two cell lines display changes of fluorescence dependent on mitochondrial energization states; 2) mitochondrial fluorescence decreases after exposure of the cells to a NO releaser; 4) the different fluorescence intensity measured under stationary growing conditions, or after activation and inhibition of constitutive NO synthase, is consistent with a steady‐state production of NO. Direct comparison of single cell fluorescence with bulk cytofluorimetry proved that the results obtained by the latter method may be misleading because of the intrinsic‐to‐measure lack of information about distribution of fluorescence within different cell compartments. The kinetic parameters describing the reactions between cytochrome oxidase, NO, and O2 may account for the puzzling (20‐fold) increase of the KM for O2 reported for cells and tissues as compared to purified cytochrome c oxidase, allowing an estimate of in vivo NO flux.—Sarti, P., Lendaro, E., Ippoliti, R., Bellelli, A., Benedetti, P. A., Brunori, M. Modulation of mitochondrial respiration by nitric oxide: investigation by single cell fluorescence microscopy. FASEB J. 13, 191–197 (1999)

Fast-reacting Thiols in Rat Hemoglobins Can Intercept Damaging Species in Erythrocytes More Efficiently Than Glutathione

The S-conjugation rates of the free-reacting thiols present on each component of rat hemoglobin with 5,5-dithio-bis(2,2-nitrobenzoic acid) (DTNB) have been studied under a variety of conditions. On the basis of their reactivity with DTNB (0.5 mm), three classes of thiols have been defined as follows: fast reacting (fHbSH), with t½ <100 ms; slow reacting (sHbSH), with t½ 30–50 s; and very slow reacting (vsHbSH), with t½ 180–270 s. Under paraphysiological conditions, fHbSH (identified with Cys-125β(H3)) conjugates with DTNB 100 times faster than glutathione and ∼4000 times more rapidly than (v)sHbSH (Cys-13α(A11) and Cys-93β(F9)). Such characteristics of fHbSH reactivity that are independent of the quaternary state of hemoglobin are mainly due to the following: (i) its low pK (∼6.9, the cysteinyl anion being stabilized by a hydrogen bond with Ser-123β(H1)) and (ii) the large exposure to the solvent (as measured by analysis of a model of the molecular surface) and make these thiols the kinetically preferred groups in rat erythrocytes for S-conjugation. In addition, because of the high cellular concentration (8 mm, i.e. four times that of glutathione), fHbSHs are expected to intercept damaging species in erythrocytes more efficiently than glutathione, thus adding a new physiopathological role (direct involvement in cellular strategies of antioxidant defense) to cysteinyl residues in proteins.

The nitrite reductase from Pseudomonas aeruginosa: Essential role of two active-site histidines in the catalytic and structural properties

Cd1 nitrite reductase catalyzes the conversion of nitrite to NO in denitrifying bacteria. Reduction of the substrate occurs at the d1-heme site, which faces on the distal side some residues thought to be essential for substrate binding and catalysis. We report the results obtained by mutating to Ala the two invariant active site histidines, His-327 and His-369, of the enzyme from Pseudomonas aeruginosa. Both mutants have lost nitrite reductase activity but maintain the ability to reduce O2 to water. Nitrite reductase activity is impaired because of the accumulation of a catalytically inactive form, possibly because the productive displacement of NO from the ferric d1-heme iron is impaired. Moreover, the two distal His play different roles in catalysis; His-369 is absolutely essential for the stability of the Michaelis complex. The structures of both mutants show (i) the new side chain in the active site, (ii) a loss of density of Tyr-10, which slipped away with the N-terminal arm, and (iii) a large topological change in the whole c-heme domain, which is displaced 20 Å from the position occupied in the wild-type enzyme. We conclude that the two invariant His play a crucial role in the activity and the structural organization of cd1 nitrite reductase from P. aeruginosa.

CO‐MP4, a polyethylene glycol‐conjugated haemoglobin derivative and carbon monoxide carrier that reduces myocardial infarct size in rats

MP4 (Hemospan) is a Hb‐based oxygen therapeutic agent, based on polyethylene‐glycol (PEG) conjugation to Hb, undergoing clinical trials as an oxygen carrier. This study describes the functional interaction between MP4 and carbon monoxide (CO), as a CO delivery agent, and the effects of CO‐MP4 on myocardial infarct size following ischaemia and reperfusion in rats.

The anti-schistosomal drug praziquantel is an adenosine antagonist

The mechanism of action of praziquantel (PZQ), the drug of choice against schistosomiasis, is still unclear. Since exposure of schistosomes to the drug is associated with calcium influx and muscular contraction, calcium channels have been suggested as the target, although direct combination of PZQ with their subunits was never demonstrated. We report a hitherto unknown effect of PZQ, namely the inhibition of nucleoside uptake, as observed in living worms using radio-isotope labelled adenosine and uridine. This effect is clearly seen in schistosomes but is absent in mammalian cells in culture. Moreover it is a specific pharmacological effect seen exclusively with the active levo-R(-)stereo isomer of the drug, and is shared by at least one benzodiazepine having antischistosomal activity. This novel effect acquires significance given that schistosomes cannot synthesize purine nucleosides de novo. A possible relationship between this novel effect and the known action of PZQ on calcium channels is discussed, since adenosine is known to bind to specific receptors and to behave as an indirect antagonist of calcium release in mammalian cells. If calcium channels were correlated with adenosine receptors also in schistosomes, as they are in mammals, this would support the hypothesis that PZQ-induced calcium influx may be correlated to adenosine receptor blockade.

A novel thermostable hemoglobin from the actinobacterium Thermobifida fusca

The gene coding for a hemoglobin‐like protein (Tf‐trHb) has been identified in the thermophilic actinobacterium Thermobifida fusca and cloned in Escherichia coli for overexpression. The crystal structure of the ferric, acetate‐bound derivative, was obtained at 2.48 Å resolution. The three‐dimensional structure of Tf‐trHb is similar to structures reported for the truncated hemoglobins from Mycobacterium tuberculosis and Bacillus subtilis in its central domain. The complete lack of diffraction patterns relative to the N‐ and C‐terminal segments indicates that these are unstructured polypeptides chains, consistent with their facile cleavage in solution. The absence of internal cavities and the presence of two water molecules between the bound acetate ion and the protein surface suggest that the mode of ligand entry is similar to that of typical hemoglobins. The protein is characterized by higher thermostability than the similar mesophilic truncated hemoglobin from B. subtilis, as demonstrated by far‐UV CD melting experiments on the cyano‐met derivatives. The ligand‐binding properties of Tf‐trHb, analyzed in stopped flow experiments, demonstrate that Tf‐trHb is capable of efficient O2 binding and release between 55 and 60 °C, the optimal growth temperature for Thermobifida fusca.

Glutathione reductase and thioredoxin reductase at the crossroad: The structure of Schistosoma mansoni thioredoxin glutathione reductase

Thioredoxin glutathione reductase (TGR) is a key flavoenzyme expressed by schistosomes that bridges two detoxification pathways crucial for the parasite survival in the hosts organism. In this article we report the crystal structure (at 2.2 Å resolution) of TGR from Schistosoma mansoni (SmTGR), deleted in the last two residues. The structure reveals the peculiar architecture of this chimeric enzyme: the small Glutaredoxin (Grx) domain at the N‐terminus is joined to the large thioredoxin reductase (TR) one via an extended complementary surface, involving residues not conserved in the Grx superfamily; the TR domain interacts with an identical partner via its C‐terminal domain, forming a dimer with a twisted “W” shape. Although lacking the penultimate Selenocysteine residue (Sec), the enzyme is still able to reduce oxidized glutathione. These data update the interpretation of the interdomain communication in TGR enzymes. The possible function of this enzyme in pathogenic parasites is discussed. Proteins 2008.

Mapping the Catalytic Cycle of Schistosoma mansoni Thioredoxin Glutathione Reductase by X-ray Crystallography

Schistosomiasis is the second most widespread human parasitic disease. It is principally treated with one drug, praziquantel, that is administered to 100 million people each year; less sensitive strains of schistosomes are emerging. One of the most appealing drug targets against schistosomiasis is thioredoxin glutathione reductase (TGR). This natural chimeric enzyme is a peculiar fusion of a glutaredoxin domain with a thioredoxin selenocysteine (U)-containing reductase domain. Selenocysteine is located on a flexible C-terminal arm that is usually disordered in the available structures of the protein and is essential for the full catalytic activity of TGR. In this study, we dissect the catalytic cycle of Schistosoma mansoni TGR by structural and functional analysis of the U597C mutant. The crystallographic data presented herein include the following: the oxidized form (at 1.9 Å resolution); the NADPH- and GSH-bound forms (2.3 and 1.9 Å, respectively); and a different crystal form of the (partially) reduced enzyme (3.1 Å), showing the physiological dimer and the entire C terminus of one subunit. Whenever possible, we determined the rate constants for the interconversion between the different oxidation states of TGR by kinetic methods. By combining the crystallographic analysis with computer modeling, we were able to throw further light on the mechanism of action of S. mansoni TGR. In particular, we hereby propose the putative functionally relevant conformational change of the C terminus after the transfer of reducing equivalents from NADPH to the redox sites of the enzyme.

The Allosteric Properties of Hemoglobin: Insights from Natural and Site Directed Mutants

After over a century of extensive research, hemoglobin has become the prototype of allosteric and cooperative proteins. Its molecular structure, known in great detail, has allowed the design of hundreds of site directed mutations, aimed at interfering with its function, and thus at testing our hypotheses on the molecular mechanisms of allostery. The wealth of information thus obtained is difficult to read except for specialists, not only because it makes use of many different technical approaches, but also because of its intrinsically patchy nature. Moreover, several researchers have tried to assign specific roles to segments of the polypeptide chains, rather than to single residues, and have tested their hypotheses by multiple point mutations or by complete replacement with the homologous segment from a different hemoglobin to produce chimeric macromolecules. This approach is in great need of a revision since putative functionally relevant segments partially overlap. This review briefly describes the structure and function of hemoglobin, and analyzes the effect of point mutations, multiple mutations and segment replacement, with special attention to possible biotechnological applications, ranging from pharmacology (Hb solutions as resuscitating fluids and sources of the protein found in hemoglobinopathies for biochemical studies) to bioreactors. Occasional reference is made to site directed mutants of myoglobin, whenever this helps clarifying perplexing results obtained on hemoglobin.