A. V. Lyashenko

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.

Purification, crystallization and preliminary X-ray study of the fungal laccase from Cerrena maxima

The crystallization and preliminary X-ray structure at 1.9 Å resolution of the fungal laccase from C. maxima are presented.

Protein crystal growth on the Russian segment of the International Space Station

Experiments on protein crystallization on the Russian segment of the International Space Station were started in 2005. These experiments were performed in the Modul’-1 protein crystallization apparatus specially designed for crystal growth by the free-interface-diffusion method. This paper describes experiments on the crystallization of lysozyme, carboxypeptidase B, and recombinant human insulin on Earth and in microgravity using the Modul’-1 protein crystallization apparatus during the ISS-11-ISS-14 space flights. Crystals of all proteins grown in microgravity have larger sizes than those grown on Earth. Space-grown crystals of lysozyme and insulin characterized by X-ray diffraction were shown to diffract to higher resolution than the Earth-grown crystals. The three-dimensional structures of Zn-insulin crystals grown both on Earth and in microgravity were established. The conformation of the Zn-insulin hexamer in the crystalline state is described.

Purification, crystallization and preliminary X-ray analysis of uridine phosphorylase from Salmonella typhimurium

The structural udp gene encoding uridine phosphorylase (UPh) was cloned from the Salmonella typhimurium chromosome and overexpressed in Escherichia coli cells. S. typhimurium UPh (StUPh) was purified to apparent homogeneity and crystallized. The primary structure of StUPh has high homology to the UPh from E. coli, but the enzymes differ substantially in substrate specificity and sensitivity to the polarity of the medium. Single crystals of StUPh were grown using hanging-drop vapor diffusion with PEG 8000 as the precipitant. X-ray diffraction data were collected to 2.9 A resolution. Preliminary analysis of the diffraction data indicated that the crystal belonged to space group P6(1(5)), with unit-cell parameters a = 92.3, c = 267.5 A. The solvent content is 37.7% assuming the presence of one StUPh hexamer per asymmetric unit.

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.

Expression, purification, crystallization and preliminary X-ray structure analysis of Vibrio cholerae uridine phosphorylase in complex with thymidine.

A high-resolution structure of the complex of Vibrio cholerae uridine phosphorylase (VchUPh) with its physiological ligand thymidine is important in order to determine the mechanism of the substrate specificity of the enzyme and for the rational design of pharmacological modulators. Here, the expression and purification of VchUPh and the crystallization of its complex with thymidine are reported. Conditions for crystallization were determined with an automated Cartesian Dispensing System using The Classics, MbClass and MbClass II Suites crystallization kits. Crystals of the VchUPh-thymidine complex (of dimensions ∼200-350 µm) were grown by the sitting-drop vapour-diffusion method in ∼7 d at 291 K. The crystallization solution consisted of 1.5 µl VchUPh (15 mg ml(-1)), 1 µl 0.1 M thymidine and 1.5 µl reservoir solution [15%(w/v) PEG 4000, 0.2 M MgCl(2).6H2O in 0.1 M Tris-HCl pH 8.5]. The crystals diffracted to 2.12 Å resolution and belonged to space group P2(1) (No. 4), with unit-cell parameters a=91.80, b=95.91, c=91.89 Å, β=119.96°. The Matthews coefficient was calculated as 2.18 Å3 Da(-1); the corresponding solvent content was 43.74%.

Purification, crystallization and preliminary X-ray structure analysis of the laccase from Ganoderma lucidum

The ligninolytic enzymes of the basidiomycetes play a key role in the global carbon cycle. A characteristic property of these enzymes is their broad substrate specificity, which has led to their use in various biotechnologies, thus stimulating research into the three-dimensional structures of ligninolytic enzymes. This paper presents the purification, crystallization and preliminary X-ray analysis of the laccase from the ligninolytic basidiomycete Ganoderma lucidum.

Comparative analysis of spatial organization of laccases from Cerrena maxima and Coriolus zonatus

Laccase (oxygen oxidoreductase, EC 1.10.3.2) belongs to the multicopper oxidase family. The main function of this enzyme is to perform electron transfer from the oxidized substrate through the mononuclear copper-containing site T1 to the oxygen molecule bound to the site T3 in the trinuclear T2/T3 cluster. The structures of two new fungal laccases from C. maxima and C. zonatus were solved on the basis of synchrotron X-ray diffraction data. Both laccases show high structural homology with laccases from other sources. The role of the carbohydrate component of laccases in structure stabilization and formation of ordered protein crystals was demonstrated. In the structures of C. maxima and C. zonatus laccases, two water channels of functional importance were found and characterized. The structural results reported in the present study characterize one of the functional states of the enzyme fixed in the crystal structure.

Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized by interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.

Atomic structure of unligated laccase from Cerrena maxima at 1.76 Å with molecular oxygen and hydrogen peroxide

The three-dimensional structure of unligated laccase from Cerrena maxima was established by X-ray diffraction at 1.76-Å resolution; Rwork = 18.07%, Rfree = 21.71%, rmsd of bond lengths, bond angles, and chiral angles are 0.008 Å, 1.19°, and 0.077°, respectively. The coordinate error for the refined structure estimated from the Luzzati plot is 0.195 Å. The maximum average error in the atomic coordinates is 0.047 Å. A total of 99.4% of amino-acid residues of the polypeptide chain are in the most favorable, allowable, and accessible regions of the Ramachandran plot. The three-dimensional structures of the complexes of laccase from C. maxima with molecular oxygen and hydrogen peroxide were determined by the molecular simulation. These data provide insight into the structural aspect of the mechanism of the enzymatic cycle. The structure factors and the refined atomic coordinates were deposited in the Protein Data Bank (PDB-ID code is 3DIV).