Andrea Ilari

The dodecameric ferritin from Listeria innocua contains a novel intersubunit iron-binding site.

Ferritin is characterized by a highly conserved architecture that comprises 24 subunits assembled into a spherical cage with 432 symmetry. The only known exception is the dodecameric ferritin from Listeria innocua. The structure of Listeria ferritin has been determined to a resolution of 2.35 Å by molecular replacement, using as a search model the structure of Dps from Escherichia coli. The Listeria 12-mer is endowed with 23 symmetry and displays the functionally relevant structural features of the ferritin 24-mer, namely the negatively charged channels along the three-fold symmetry axes that serve for iron entry into the cavity and a negatively charged internal cavity for iron deposition. The electron density map shows 12 iron ions on the inner surface of the hollow core, at the interface between monomers related by two-fold axes. Analysis of the nature and stereochemistry of the iron-binding ligands reveals strong similarities with known ferroxidase sites. The L. innocua ferritin site, however, is the first described so far that has ligands belonging to two different subunits and is not contained within a four-helix bundle.

Iron and proteins for iron storage and detoxification

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.

Iron Incorporation into Escherichia coli Dps Gives Rise to a Ferritin-like Microcrystalline Core

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 3d5 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, witht 1 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.

The X-ray structure of ferric Escherichia coli flavohemoglobin reveals an unexpected geometry of the distal heme pocket

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 anR-factor of 19%. The overall fold is similar to that of ferrous lipid-bound Alcaligenes eutrophusflavohemoglobin 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.

Polyamine metabolism in Leishmania: from arginine to trypanothione

Polyamines (PAs) are essential metabolites in eukaryotes, participating in a variety of proliferative processes, and in trypanosomatid protozoa play an additional role in the synthesis of the critical thiol trypanothione. The PAs are synthesized by a metabolic process which involves arginase (ARG), which catalyzes the enzymatic hydrolysis of l-arginine (l-Arg) to l-ornithine and urea, and ornithine decarboxylase (ODC), which catalyzes the enzymatic decarboxylation of l-ornithine in putrescine. The S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the irreversible decarboxylation of S-adenosylmethionine (AdoMet), generating the decarboxylated S-adenosylmethionine (dAdoMet), which is a substrate, together with putrescine, for spermidine synthase (SpdS). Leishmania parasites and all the other members of the trypanosomatid family depend on spermidine for growth and survival. They can synthesize PAs and polyamine precursors, and also scavenge them from the microenvironment, using specific transporters. In addition, Trypanosomatids have a unique thiol-based metabolism, in which trypanothione (N1-N8-bis(glutathionyl)spermidine, T(SH)2) and trypanothione reductase (TR) replace many of the antioxidant and metabolic functions of the glutathione/glutathione reductase (GR) and thioredoxin/thioredoxin reductase (TrxR) systems present in the host. Trypanothione synthetase (TryS) and TR are necessary for the protozoa survival. Consequently, enzymes involved in spermidine synthesis and its utilization, i.e. ARG, ODC, AdoMetDC, SpdS and, in particular, TryS and TR, are promising targets for drug development.

Silver Ion Incorporation and Nanoparticle Formation Inside the Cavity of Pyrococcus Furiosus Ferritin: Structural and Size-Distribution Analyses.

Highly symmetrical protein cage architectures from three different iron storage proteins, heavy and light human ferritin chains (HuHFt and HuLFt) and ferritin from the hyperthemophilic bacterium Pyrococcus furiosus (PfFt), have been used as models for understanding the molecular basis of silver ion deposition and metal core formation inside the protein cavity. Biomineralization using protein cavities is an important issue for the fabrication of biometamaterials under mild synthetic conditions. Silver nanoparticles (AgNPs) were produced with high yields within PfFt but not within HuHFt and HuLFt. To explain the molecular basis of silver incorporation, the X-ray crystal structure of Ag-containing PfFt has been solved. This is the first structure of a silver containing ferritin reported to date, and it revealed the presence of specific binding and nucleation sites of Ag(I) that are not conserved in other ferritin templates. The AgNP encapsulated by PfFt were further characterized by the combined use of different physical-chemical techniques. These showed that the AgNPs are endowed with a narrow size distribution (2.1 +/- 0.4 nm), high stability in water solution at millimolar concentration, and high thermal stability. These properties make the AgNP obtained within PftFt exploitable for a range of applications, in fields as diverse as catalysis in water, preparation of metamaterials, and in vivo diagnosis and antibacterial or tumor therapy.

Structural Basis of Enzymatic S-Norcoclaurine Biosynthesis.

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 2.1Å 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.

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.

Protein structure determination by x-ray crystallography.

X-ray biocrystallography is the most powerful method to obtain a macromolecular structure. The improvement of computational technologies in recent years and the development of new and powerful computer programs together with the enormous increment in the number of protein structures deposited in the Protein Data Bank, render the resolution of new structures easier than in the past. The aim of this chapter is to provide practical procedures useful for solving a new structure. It is impossible to give more than a flavor of what the x-ray crystallographic technique entails in one brief chapter; therefore, this chapter focuses its attention on the Molecular Replacement method. Whenever applicable, this method allows the resolution of macromolecular structures starting from a single data set and a search model downloaded from the PDB, with the aid only of computer work.

Antioxidant Dps protein from the thermophilic cyanobacterium Thermosynechococcus elongatus.

DNA‐binding proteins from starved cells (Dps proteins) protect bacteria primarily from oxidative damage. They are composed of 12 identical subunits assembled with 23‐symmetry to form a compact cage‐like structure known to be stable at temperatures > 70 °C and over a wide pH range. Thermosynechococcus elongatus Dps thermostability is increased dramatically relative to mesophilic Dps proteins. Hydrophobic interactions at the dimeric and trimeric interfaces called Dps‐like are replaced by salt bridges and hydrogen bonds, a common strategy in thermophiles. Moreover, the buried surface area at the least‐extended Dps‐like interface is significantly increased. A peculiarity of T. elongatus Dps is the presence of a chloride ion coordinated with threefold symmetry‐related arginine residues lining the opening of the Dps‐like pore toward the internal cavity. T. elongatus Dps conserves the unusual intersubunit ferroxidase centre that allows the Dps protein family to oxidize Fe(II) with hydrogen peroxide, thereby inhibiting free radical production via Fenton chemistry. This catalytic property is of special importance in T. elongatus (which lacks the catalase gene) in the protection of DNA and photosystems I and II from hydrogen peroxide‐mediated oxidative damage.