N. E. Zhukhlistova

Three-dimensional organization of three-domain copper oxidases: A review

Abstract“Blue” copper-containing proteins are multidomain proteins that utilize a unique redox property of copper ions. Among other blue multicopper oxidases, three-domain oxidases belong to the group of proteins that exhibit a wide variety of compositions in amino acid sequences, functions, and occurrences in organisms. This paper presents a review of the data obtained from X-ray diffraction investigations of the three-dimensional structures of three-domain multicopper oxidases, such as the ascorbate oxidase catalyzing oxidation of ascorbate to dehydroascorbate and its three derivatives; the multicopper oxidase CueO (the laccase homologue); the laccases isolated from the basidiomycetes Coprinus cinereus, Trametes versicolor, Coriolus zonatus, Cerrena maxima, and Rigidoporus lignosus and the ascomycete Melanocarpus albomyces; and the bacterial laccases CotA from the endospore coats of Bacillus subtilis. A comparison of the molecular structures of the laccases of different origins demonstrates that, structurally, these objects are highly conservative. This obviously indicates that the catalytic activity of the enzymes under consideration is characterized by similar mechanisms.

Three-Dimensional Structure of Laccase from Coriolus zonatus at 2.6 Å Resolution

Laccase (oxygen oxidoreductase, EC 1.14.18.1) belongs to the copper-containing oxidases. This enzyme catalyzes reduction of molecular oxygen by different organic and inorganic compounds to water without the formation of hydrogen peroxide. The three-dimensional structure of native laccase from Coriolus zonatus was solved and refined at 2.6 Å resolution (Rfactor = 21.23%, Rfree = 23.82%, rms deviations for the bond lengths and bond angles are 0.008 Å and 1.19°, respectively). The primary structure of the polypeptide chain and the architecture of the active site were refined. The carbohydrate component of the enzyme was identified. The access and exit water channels providing the access of molecular oxygen to the active site and release of water, which is the reduction product of molecular oxygen, from the protein molecule were found in the structure.

Three-dimensional structure of thymidine phosphorylase from E. coli in complex with 3′-azido-2′-fluoro-2′,3′-dideoxyuridine

The three-dimensional structures of thymidine phosphorylase from E. coli containing the bound sulfate ion in the phosphate-binding site and of the complex of thymidine phosphorylase with sulfate in the phosphate-binding site and the inhibitor 3′-azido-2′-fluoro-2′,3′-dideoxyuridine (N3F-ddU) in the nucleoside-binding site were determined at 1.55 and 1.50 Å resolution, respectively. The amino-acid residues involved in the ligand binding and the hydrogen-bond network in the active site occupied by a large number of bound water molecules are described. A comparison of the structure of thymidine phosphorylase in complex with N3F-ddU with the structure of pyrimidine nucleoside phosphorylase from St. Aureus in complex with the natural substrate thymidine (PDB_ID: 3H5Q) shows that the substrate and the inhibitor in the nucleoside-binding pocket have different orientations. It is suggested that the position of N3F-ddU can be influenced by the presence of the azido group, which prefers a hydrophobic environment. In both structures, the active sites of the subunits are in the open conformation.

Comparative analysis of three-dimensional structures of homodimers of uridine phosphorylase from Salmonella typhimurium in the unligated state and in a complex with potassium ion

The spatial organization of the homodimer of unligated uridine phosphorylase from Salmonella typhimurium (St UPh) was determined with high accuracy. The structure was refined at 1.80 Å resolution to Rwork = 16.1% and Rfree = 20.0%. The rms deviations for the bond lengths, bond angles, and chiral angles are 0.006 Å, 1.042°, and 0.071°, respectively. The coordinate error estimated by the Luzzati plot is 0.166 Å. The coordinate error based on the maximum likelihood is 0.199 Å. A comparative analysis of the spatial organization of the homodimer in two independently refined structures and the structure of the homodimer St UPh in the complex with a K+ ion was performed. The substrate-binding sites in the homodimers StUPhs in the unligated state were found to act asynchronously. In the presence of a potassium ion, the three-dimensional structures of the subunits in the homodimer are virtually identical, which is apparently of importance for the synchronous action of both substrate-binding sites. The atomic coordinates of the refined structure of the homodimer and structure factors have been deposited in the Protein Data Bank (PDB ID code 3DPS).

X-ray crystal structure of the complex of enniatin B with KNCS

The crystal structure of the complex of enniatin B, cyclo[-(L-MeVal-D-Hyi)3-](C33H57N3O9), with KNCS is determined by X-ray diffraction [CuKαradiation, R = 0.0594 for 7925 reflections with I > 2 σ(I)]. The crystals belong to the space group P3, a = 24.448(5) Å, c = 23.578(5) Å, V = 12277(9) Å3, and Z = 6. The unit cell contains 12 symmetrically independent molecules of the antibiotic, which are located on crystallographic threefold axes. The K+ ions are located on the threefold axes and are coordinated by the carbonyl oxygen atoms of the hydroxy acid or amino acid residues of the enniatin molecules to form prisms, twisted prisms, and antiprisms. All the independent enniatin molecules retain the principal conformational features revealed earlier in the structures of enniatin B and its complexes.

Three-dimensional structure of phosphoribosyl pyrophosphate synthetase from E. coli at 2.71 Å resolution

Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystallized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was collected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homologous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and regulatory sites were shown to be identical in both proteins.

Uridine phosphorylase in biomedical, structural, and functional aspects: A review

The activation of xenobiotics often causes malignant tumor cells to resist chemotherapeutic treatment. Uridine phosphorylase is the key enzyme of pyrimidine metabolism and catalyzes the reversible phosphorylation of uridine with the formation of uracil and ribose-1-phosphate. High-selectivity anticancer agents based on uridine phosphorylase inhibitors are promising for treating both oncological and infection diseases. New medicinal preparations can be predicted and rationally developed only on the basis of detailed biomedical, structural, and functional knowledge about the biomacromolecular target enzyme-drug complex.

Three-dimensional structure of E. Coli purine nucleoside phosphorylase at 0.99 Å resolution

Purine nucleoside phosphorylases (PNPs) catalyze the reversible phosphorolysis of nucleosides and are key enzymes involved in nucleotide metabolism. They are essential for normal cell function and can catalyze the transglycosylation. Crystals of E. coli PNP were grown in microgravity by the capillary counterdiffusion method through a gel layer. The three-dimensional structure of the enzyme was determined by the molecular-replacement method at 0.99 Å resolution. The structural features are considered, and the structure of E. coli PNP is compared with the structures of the free enzyme and its complexes with purine base derivatives established earlier. A comparison of the environment of the purine base in the complex of PNP with formycin A and of the pyrimidine base in the complex of uridine phosphorylase with thymidine revealed the main structural features of the base-binding sites. Coordinates of the atomic model determined with high accuracy were deposited in the Protein Data Bank (PDB_ID: 4RJ2).

Crystallization and preliminary X-ray diffraction study of phosphoribosyl pyrophosphate synthetase from E. Coli

Enzymes of the phosphoribosyl pyrophosphate synthetase family (PRPPS, EC 2.7.6.1) catalyze the formation of 5-phosphoribosyl pyrophosphate (5-PRPP) from adenosine triphosphate and ribose 5-phosphate. 5-Phosphoribosyl pyrophosphate is an important intermediate in the synthesis of purine, pyrimidine, and pyridine nucleotides, as well as of the amino acids histidine and tryptophan. The crystallization conditions for E. coli PRPPS were found by the vapor-diffusion technique and were optimized to apply the capillary counter-diffusion technique. The X-ray diffraction data set was collected from the crystals grown by the counter-diffusion technique using a synchrotron radiation source to 3.1-Å resolution. The crystals of PRPPS belong to sp. gr. P6322 and have the following unit-cell parameters: a = b = 104.44 Å, c = 124.98 Å, α = β = 90°, γ = 120°. The collected X-ray diffraction data set is suitable for the solution of the three-dimensional structure of PRPPS at 3.1-Å resolution.

Structural basis for the mechanism of inhibition of uridine phosphorylase from Salmonella typhimurium

The three-dimensional structures of three complexes of Salmonella typhimurium uridine phosphorylase with the inhibitor 2,2′-anhydrouridine, the substrate PO4, and with both the inhibitor 2,2′-anhydrouridine and the substrate PO4 (a binary complex) were studied in detail by X-ray diffraction. The structures of the complexes were refined at 2.38, 1.5, and 1.75 Å resolution, respectively. Changes in the three-dimensional structure of the subunits in different crystal structures are considered depending on the presence or absence of the inhibitor molecule and (or) the phosphate ion in the active site of the enzyme. The presence of the phosphate ion in the phosphate-binding site was found to substantially change the orientations of the side chains of the amino-acid residues Arg30, Arg91, and Arg48 coordinated to this ion. A comparison showed that the highly flexible loop L9 is unstable. The atomic coordinates of the refined structures of the complexes and the corresponding structure factors were deposited in the Protein Data Bank (their PDB ID codes are 3DD0 and 3C74). The experimental data on the spatial reorganization of the active site caused by changes in its functional state from the unligated to the completely inhibited state suggest the structural basis for the mechanism of inhibition of Salmonella typhimurium uridine phosphorylase.