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Inositol polyphosphate 1-phosphatase from Entamoeba histolytica (EhIPPase) is an Mg(2+)-dependent and Li(+)-sensitive enzyme that catalyzes the hydrolysis of inositol 1,4-bisphosphate [Ins(1,4)P2] into myo-inositol 1-monophosphate and PO4(3-). In the present work, EhIPPase has been biochemically identified and its crystal structure has been determined in the presence of Mg(2+) and PO4(3-) at 2.5 Å resolution. This enzyme was previously classified as a 3'(2'),5'-bisphosphate nucleotidase in the NCBI, but its biochemical activity and structural analysis suggest that this enzyme behaves more like an inositol polyphosphate 1-phosphatase. The ability of EhIPPase to hydrolyze the smaller Ins(1,4)P2 better than the bulkier 3'-phosphoadenosine 5'-phosphate (PAP) is explained on the basis of the orientations of amino-acid residues in the binding site. This structure is the first of its class to be determined from any protozoan parasite, and is the third to determined among all organisms, following its rat and bovine homologues. The three-dimensional fold of EhIPPase is similar to those of other members of the inositol monophosphatase superfamily, which also includes inositol monophosphatase, 3'(2'),5'-bisphosphate nucleotidase and fructose-1,6-bisphosphate 1-phosphatase. They all share conserved residues essential for metal binding and substrate hydrolysis, with the motif D-Xn-EE-Xn-DP(I/L)DG(S/T)-Xn-WD-Xn-GG. The structure is divided into two domains, namely α+β and α/β, and the substrate and metal ions bind between them. However, the ability of each enzyme class to act specifically on its cognate substrate is governed by the class-specific amino-acid residues at the active site.

Staphylococcus aureus TenA (SaTenA) is a thiaminase type II enzyme that catalyzes the deamination of aminopyrimidine, as well as the cleavage of thiamine into 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) and 5-(2-hydroxyethyl)-4-methylthiazole (THZ), within thiamine (vitamin B1) metabolism. Further, by analogy with studies of Bacillus subtilis TenA, SaTenA may act as a regulator controlling the secretion of extracellular proteases such as the subtilisin type of enzymes in bacteria. Thiamine biosynthesis has been identified as a potential drug target of the multi-resistant pathogen S. aureus and therefore all enzymes involved in the S. aureus thiamine pathway are presently being investigated in detail. Here, the structure of SaTenA, determined by molecular replacement and refined at 2.7 Å resolution to an R factor of 21.6% with one homotetramer in the asymmetric unit in the orthorhombic space group P212121, is presented. The tetrameric state of wild-type (WT) SaTenA was postulated to be the functional biological unit and was confirmed by small-angle X-ray scattering (SAXS) experiments in solution. To obtain insights into structural and functional features of the oligomeric SaTenA, comparative kinetic investigations as well as experiments analyzing the structural stability of the WT SaTenA tetramer versus a monomeric SaTenA mutant were performed.

The crystal structure of the myotoxic, cell-penetrating, basic polypeptide crotamine isolated from the venom of Crotalus durissus terrificus has been determined by single-wavelength anomalous dispersion techniques and refined at 1.7 Å resolution. The structure reveals distinct cationic and hydrophobic surface regions that are located on opposite sides of the molecule. This surface-charge distribution indicates its possible mode of interaction with negatively charged phospholipids and other molecular targets to account for its diverse pharmacological activities. Although the sequence identity between crotamine and human β-defensins is low, the three-dimensional structures of these functionally related peptides are similar. Since crotamine is a leading member of a large family of myotoxic peptides, its structure will provide a basis for the design of novel cell-penetrating molecules.

Uridine phosphorylase (UPh), which is a key enzyme in the reutilization pathway of pyrimidine nucleoside metabolism, is a validated target for the treatment of infectious diseases and cancer. A detailed analysis of the interactions of UPh with the therapeutic ligand 5-fluorouracil (5-FUra) is important for the rational design of pharmacological inhibitors of these enzymes in prokaryotes and eukaryotes. Expanding on the preliminary analysis of the spatial organization of the active centre of UPh from the pathogenic bacterium Salmonella typhimurium (StUPh) in complex with 5-FUra [Lashkov et al. (2009), Acta Cryst. F65, 601-603], the X-ray structure of the StUPh-5-FUra complex was analysed at atomic resolution and an in silico model of the complex formed by the drug with UPh from Vibrio cholerae (VchUPh) was generated. These results should be considered in the design of selective inhibitors of UPhs from various species.

Uridine nucleoside phosphorylase is an important drug target for the development of anti-infective and antitumour agents. The X-ray crystal structure of Salmonella typhimurium uridine nucleoside phosphorylase (StUPh) complexed with its inhibitor 2,2'-anhydrouridine, phosphate and potassium ions has been solved and refined at 1.86 A resolution (R(cryst) = 17.6%, R(free) = 20.6%). The complex of human uridine phosphorylase I (HUPhI) with 2,2'-anhydrouridine was modelled using a computational approach. The model allowed the identification of atomic groups in 2,2'-anhydrouridine that might improve the interaction of future inhibitors with StUPh and HUPhI.

The crystal structure of a serine-specific tRNA acceptor-stem microhelix, the binding site for the seryl-tRNA synthetase, was solved by X-ray analysis. This seven-base-pair tRNA(Ser) microhelix forms endless rows of helices in the crystal lattice, with two helices stacking 'head-to-head' onto each other, resulting in an intermolecular guanosine stacking of the first purine nucleotides at the 5'-strands of the tRNA(Ser) microhelices. A network of 75 water loci could be associated with each RNA duplex. Unusual local geometric backbone parameters could be detected in the region of the G4 phosphate located in the 5'-strand of the helix, which lead to a ;kink' in this region and to an irregularly bent helix. The role of the specific hydration pattern and of the irregular conformation of the tRNA(Ser) acceptor-stem helix is discussed and summarized.

The presynaptic viperotoxin F is the major lethal component of the venom of Vipera russelli formosensis (Taiwan viper). It is a heterodimer of two highly homologous (65% identity) but oppositely charged subunits: a basic and neurotoxic PLA(2) (RV-4) and an acidic non-toxic component with a very low enzymatic activity (RV-7). The crystal structure of the complex has been determined by molecular replacement and refined to 1.9 A resolution and an R factor of 22.3% with four RV-4/RV-7 complexes in the asymmetric unit, which do not exhibit any local point-group symmetry. The complex formation decreases the accessible surface area of the two subunits by approximately 1425 A(2). Both PLA(2)s are predicted to have very low, if any, anticoagulant activity. The structure of viperotoxin F is compared with that of the heterodimeric neurotoxin vipoxin from the venom of another viper, V. ammodytes meridionalis. The structural basis for the differences between the pharmacological activities of the two toxins is discussed. The neutralization of the negative charge of the major ligand for Ca(2+), Asp49, by intersubunit salt bridges is probably a common mechanism of self-stabilization of heterodimeric Viperinae snake-venom neurotoxins in the absence of bound calcium.