. These results and in vitro DNA damage assays indicate that the protective effect of Dps on DNA most likely is exerted through a dual action, the physical association with DNA and the ability to nullify the toxic combination of Fe(II) and H 2 O 2 . In the latter process a hydrous ferric oxide mineral core is produced within the protein, thus avoiding oxidative damage mediated by Fenton chemistry.

Leishmaniasis is a disease that affects 2 million people and kills 70000 persons every year. It is caused by Leishmania species, which are human protozoan parasites of the trypanosomatidae family. Trypanosomatidae differ from the other eukaryotes in their specific redox metabolism because the glutathione/glutathione reductase system is replaced by the unique trypanothione/trypanothione reductase system. The current treatment of leishmaniasis relies mainly on antimonial drugs. The crystal structures of oxidized trypanothione reductase (TR) from Leishmania infantum and of the complex of reduced TR with NADPH and Sb(III), reported in this paper, disclose for the first time the molecular mechanism of action of antimonial drugs against the parasite. Sb(III), which is coordinated by the two redox-active catalytic cysteine residues (Cys52 and Cys57), one threonine residue (Thr335), and His461' of the 2-fold symmetry related subunit in the dimer, strongly inhibits TR activity. Because TR is essential for the parasite survival and virulence and it is absent in mammalian cells, these findings provide insights toward the design of new more affordable and less toxic drugs against Leishmaniasis.

The enzyme norcoclaurine synthase (NCS) catalyzes the stereospecific Pictet-Spengler cyclization between dopamine and 4-hydroxyphenylacetaldehyde, the key step in the benzylisoquinoline alkaloid biosynthetic pathway. The crystallographic structure of norcoclaurine synthase from Thalictrum flavum in its complex with dopamine substrate and the nonreactive substrate analogue 4-hydroxybenzaldehyde has been solved at 2.1 Å resolution. NCS shares no common features with the functionally correlated "Pictet-Spenglerases" that catalyze the first step of the indole alkaloids pathways and conforms to the overall fold of the Bet v1-like protein. The active site of NCS is located within a 20-Å -long catalytic tunnel and is shaped by the side chains of a tyrosine, a lysine, an aspartic, and a glutamic acid. The geometry of the amino acid side chains with respect to the substrates reveals the structural determinants that govern the mechanism of the stereoselective Pictet-Spengler cyclization, thus establishing an excellent foundation for the understanding of the finer details of the catalytic process. Site-directed mutations of the relevant residues confirm the assignment based on crystallographic findings.

Escherichia coli Dps belongs to a family of bacterial stress-induced proteins to protect DNA from oxidative damage. It shares with Listeria innocua ferritin several structural features, such as the quaternary assemblage and the presence of an unusual ferroxidase center. Indeed, it was recently recognized to be able to oxidize and incorporate iron. Since ferritins are endowed with the unique capacity to direct iron deposition toward formation of a microcrystalline core, the structure of iron deposited in the E. coli Dps cavity was studied. Polarized single crystal absorption microspectrophotometry of iron-loaded Dps shows that iron ions are oriented. The spectral properties in the high spin 3d 5 configuration point to a crystal form with tetrahedral symmetry where the tetrahedron center is occupied by iron ions and the vertices by oxygen. Crystals of iron-loaded Dps also show that, as in mammalian ferritins, iron does not remain bound to the site after oxidation has taken place. The kinetics of the iron reduction/release process induced by dithionite were measured in the crystal and in solution. The reaction appears to have two phases, with t1 ⁄2 of a few seconds and several minutes at neutral pH values, as in canonical ferritins. This behavior is attributed to a similar composition of the iron core.

Auranofin is a gold(I)-containing drug in clinical use as an antiarthritic agent. Recent studies showed that auranofin manifests interesting antiparasitic actions very likely arising from inhibition of parasitic enzymes involved in the control of the redox metabolism. Trypanothione reductase is a key enzyme of Leishmania infantum polyamine-dependent redox metabolism, and a validated target for antileishmanial drugs. As trypanothione reductase contains a dithiol motif at its active site and gold(I) compounds are known to be highly thiophilic, we explored whether auranofin might behave as an effective enzyme inhibitor and as a potential antileishmanial agent. Notably, enzymatic assays revealed that auranofin causes indeed a pronounced enzyme inhibition. To gain a deeper insight into the molecular basis of enzyme inhibition, crystals of the auranofin-bound enzyme, in the presence of NADPH, were prepared, and the X-ray crystal structure of the auranofin–trypanothione reductase–NADPH complex was solved at 3.5 Å resolution. In spite of the rather low resolution, these data were of sufficient quality as to identify the presence of the gold center and of the thiosugar of auranofin, and to locate them within the overall protein structure. Gold binds to the two active site cysteine residues of TR, i.e. Cys52 and Cys57, while the thiosugar moiety of auranofin binds to the trypanothione binding site; thus auranofin appears to inhibit TR through a dual mechanism. Auranofin kills the promastigote stage of L. infantum at micromolar concentration; these findings will contribute to the design of new drugs against leishmaniasis.

Agrobacterium tumefaciens Dps (DNA-binding proteins from starved cells), encoded by the dps gene located on the circular chromosome of this plant pathogen, was cloned, and its structural and functional properties were determined in vitro. In Escherichia coli Dps, the family prototype, the DNA binding properties are thought to be associated with the presence of the lysine-containing N-terminal tail that extends from the protein surface into the solvent. The x-ray crystal structure of A. tumefaciens Dps shows that the positively charged N-terminal tail, which is 11 amino acids shorter than in the E. coli protein, is blocked onto the protein surface. This feature accounts for the lack of interaction with DNA. The intersubunit ferroxidase center characteristic of Dps proteins is conserved and confers to the A. tumefaciens protein a ferritin-like activity that manifests itself in the capacity to oxidize and incorporate iron in the internal cavity and to release it after reduction. In turn, sequestration of Fe(II) correlates with the capacity of A. tumefaciens Dps to reduce the production of hydroxyl radicals from H 2 O 2 through Fenton chemistry. These data demonstrate conclusively that DNA protection from oxidative damage in vitro does not require formation of a Dps-DNA complex. In vivo, the hydroxyl radical scavenging activity of A. tumefaciens Dps may be envisaged to act in concert with catalase A to counteract the toxic effect of H 2 O 2 , the major component of the plant defense system when challenged by the bacterium.

The x-ray structure of ferric unliganded lipid-free Escherichia coli flavohemoglobin has been solved to a resolution of 2.2 Å and refined to an R-factor of 19%. The overall fold is similar to that of ferrous lipid-bound Alcaligenes eutrophus flavohemoglobin with the notable exception of the E helix positioning within the globin domain and a rotation of the NAD binding module with respect to the FAD-binding domain accompanied by a substantial rearrangement of the C-terminal region. An inspection of the heme environment in E. coli flavohemoglobin reveals an unexpected architecture of the distal pocket. In fact, the distal site is occupied by the isopropyl side chain Leu-E11 that shields the heme iron from the residues in the topological positions predicted to interact with heme iron-bound ligands, namely Tyr-B10 and Gln-E7, and stabilizes a pentacoordinate ferric iron species. Ligand binding properties are consistent with the presence of a pentacoordinate species in solution as indicated by a very fast second order combination rates with imidazole and azide. Surprisingly, imidazole, cyanide, and azide binding profiles at equilibrium are not accounted for by a single site titration curve but are biphasic and strongly suggest the presence of two distinct conformers within the liganded species.Flavohemoglobins are oxygen-binding proteins composed of a typical globin domain containing a B-type heme fused with a ferredoxin reductase-like FAD-and NAD-binding domain. Together with single domain bacterial hemoglobins and six-helices containing "truncated hemoglobins," flavohemoglobins are part of a vast family of "hemoglobin-like" proteins whose functions are still elusive. Because of the identification of flavohemoglobin genes in a wide variety of prokaryotic and eukaryotic microorganisms, a crescendo of experimental observations has revealed that these proteins possess unique structural and functional properties unrelated to those of hemoproteins involved in oxygen transport and storage (1).Escherichia coli flavohemoglobin (HMP) 1 is certainly the most extensively studied protein within the bacterial hemoglobin family. At present, the most credited physiological function of HMP has been inferred from the observation that protein expression in the bacterial cell is enhanced in the presence of nitric oxide releasers in the culture medium (2-4). Accordingly, HMP has been shown to be able to catalyze the oxidation of free nitric oxide to nitrate both in vivo and in vitro in the presence of oxygen and NADH (5). In this framework, HMP is thought to be involved in the response of the bacterial cell to the potentially harmful role of free nitric oxide in giving rise to nitrosative stress. Nevertheless, although the nitric-oxide dioxygenase activity stands as the major functional hypothesis for HMP, the molecular mechanism and the structural determinants at the basis of this enzymic function remain poorly understood. In particular, it has not been established as yet how the amino acid side chains in the distal heme pocket affect...

Iron is required by most organisms, but is potentially toxic due to the low solubility of the stable oxidation state, Fe(III), and to the tendency to potentiate the production of reactive oxygen species, ROS. The reactivity of iron is counteracted by bacteria with the same strategies employed by the host, namely by sequestering the metal into ferritin, the ubiquitous iron storage protein. Ferritins are highly conserved, hollow spheres constructed from 24 subunits that are endowed with ferroxidase activity and can harbour up to 4500 iron atoms as oxy-hydroxide micelles. The release of the metal upon reduction can alter the microorganism-host iron balance and hence permit bacteria to overcome iron limitation. In bacteria, the relevance of the Dps (DNA-binding proteins from starved cells) family in iron storage-detoxification has been recognized recently. The seminal studies on the protein from Listeria innocua demonstrated that Dps proteins have ferritin-like activity and most importantly have the capacity to attenuate the production of ROS. This latter function allows bacterial pathogens that lack catalase, e.g. Porphyromonas gingivalis, to survive in an aerobic environment and resist to peroxide stress.