Single-point substitution F97M leads to in cellulo crystallization of the biphotochromic protein moxSAASoti.

To enhance the photoconversion performance of biphotochromic moxSAASoti protein, a substitution F97 M was introduced. In addition to enhancing the target properties, this substitution also resulted in the crystallization of the recombinant protein within living HeLa cells (also referred to as in cellulo crystallization). The phenomenon of protein crystallization in living cells is not unique, yet the mechanisms and application of in cellulo crystallization remain significant for further research. However, in cellulo crystallization is atypical for fluorescent proteins and detrimental for their biotechnological application. The objective of this study was to elucidate the underlying mechanisms responsible for the crystallization of moxSAASotiF97Min cellulo. For this purpose, the crystal structure of the green form of biphotochromic protein moxSAASotiF97M was determined at high resolution, which surprisingly has a space group, different from those of parent mSAASotiC21N. The analysis provided allowed to propose a mechanism of new crystal contacts formation, which might be a cause of in cellulo protein crystallization.

Highly-Active Recombinant Formate Dehydrogenase from Pathogenic Bacterium Staphylococcus aureus: Preparation and Crystallization.

# These authors contributed equally to the work. NAD+-dependent formate dehydrogenase from Staphylococcus aureus (SauFDH) is one of the key enzymes responsible for the survival of this pathogen in the form of biofilms. 3D structure of the enzyme might be helpful in the search for highly specific SauFDH inhibitors that can be used as antibacterial agents exactly against S. aureus biofilms. Here, we prepared a recombinant SauFDH in Escherichia coli cells with a yield of 1 g target protein per liter medium. The developed procedure for the enzyme purification allowed to obtain 400 mg of homogenous enzyme with 61% yield. The specific activity of the purified recombinant SauFDH was 20 U per mg protein, which was 2 times higher than the previously reported activities of formate dehydrogenases. We also found crystallization conditions in the course of two rounds of optimization and obtained 200- and 40-µm crystals for the SauFDH apo- and holoenzymes, respectively. X-ray analysis using synchrotron X-ray sources produced diffraction data sufficient for solving the three-dimensional structures of the apo- and holoenzymes with the resolution of 2.2 and 2.7 Å, respectively. Crystals of the apo- and holoforms of SauFDH had different crystal space groups, which suggest coenzyme binding in the SauFDH holoenzyme.

Expression, purification and biophysical characterization of recombinant Streptomyces violaceoruber phospholipase PLA2 overproduced in Pichia pastoris.

Aim: The main purpose of this work was to develop new protocols for high yield purification of secretory phospholipase A2 (PLA2) and to investigate its biophysical properties.Materials and methods: We have used a Pichia pastoris expression system for PLA2 expression and two-stage chromatography for its purification. The biophysical properties of PLA2 were investigated by circular dichroism.Results: A scalable method for high yield purification of recombinant Streptomyces violaceruber PLA2 was developed. The PLA2 from S. violaceruber was expressed in the methylotrophic yeast P. pastoris. Functional active phospholipase A2 with specific activity 73 U/mg was purified with a concentration of at least 3 mg/mL. The role of different divalent ions in PLA2 thermostability were evaluated. Ca2+ and Ba2+ ions significantly increased thermostability of the enzyme.

Effect of Ketosubstrate on the Product Yield in the Transamination Reaction Catalyzed by Transaminase from Thermoproteus uzoniensis.

The study of the equilibrium of reactions catalyzed by thermostable enzymes is in demand for the development of biotechnological enzyme processes. The results of the analysis of equilibrium of transamination reaction catalyzed by thermostable transaminase from the archaeon Thermoproteus uzoniensis are presented below. A comparison of the conversion of substrates was performed for reactions with L-leucine and pyruvate and L-leucine and 2-oxobutyrate at 65°C. The establishment of the equilibrium was controlled by a decrease in the concentration of 2-oxobutyrate or pyruvate and by the accumulation of the keto analog of L-leucine. It was shown that the degree of conversion of L-leucine in the reaction with specific 2-oxobutyrate is higher than in the reaction with nonspecific pyruvate.

The Influence of Myofibrils on the Proliferation and Differentiation of Myoblasts Cocultured with Macrophages.

We studied the effect of myofibrils on proliferation and differentiation of myoblasts cocultured with macrophages as well as the effect of incubation of macrophages with myofibrils on the expression by macrophages of the compounds that are cytokines for muscle cells. In the cocultures, macrophages stimulated the proliferation of myoblasts. Myofibrils greatly enhanced the stimulating effect of macrophages, whereas lipopolysaccharide (LPS) completely abolished it. The culture medium conditioned by macrophages activated the proliferation of myoblasts that were incubated with myofibrils but inhibited it when myoblasts were incubated with LPS. Possibly, myofibrils and their constituent proteins activate macrophages in an alternative pathway, enriching the population with M2-type macrophages.Z.

Properties of Bacterial and Archaeal Branched-Chain Amino Acid Aminotransferases.

Branched-chain amino acid aminotransferases (BCATs) catalyze reversible stereoselective transamination of branched-chain amino acids (BCAAs) L-leucine, L-isoleucine, and L-valine. BCATs are the key enzymes of BCAA metabolism in all organisms. The catalysis proceeds through the ping-pong mechanism with the assistance of the cofactor pyridoxal 5'-phosphate (PLP). BCATs differ from other (S)-selective transaminases (TAs) in 3D-structure and organization of the PLP-binding domain. Unlike other (S)-selective TAs, BCATs belong to the PLP fold type IV and are characterized by the proton transfer on the re-face of PLP, in contrast to the si-specificity of proton transfer in fold type I (S)-selective TAs. Moreover, BCATs are the only (S)-selective enzymes within fold type IV TAs. Dual substrate recognition in BCATs is implemented via the "lock and key" mechanism without side-chain rearrangements of the active site residues. Another feature of the active site organization in BCATs is the binding of the substrate α-COOH group on the P-side of the active site near the PLP phosphate group. Close localization of two charged groups seems to increase the effectiveness of external aldimine formation in BCAT catalysis. In this review, the structure-function features and the substrate specificity of bacterial and archaeal BCATs are analyzed. These BCATs differ from eukaryotic ones in the wide substrate specificity, optimal temperature, and reactivity toward pyruvate as the second substrate. The prospects of biotechnological application of BCATs in stereoselective synthesis are discussed.

[Peroxidase activity of octaheme nitrite reductases from bacteria of the Thioalkalivibrio genus].

Closely related penta- and octaheme nitrite reductases catalyze the reduction of nitrite, nitric oxide, and hydroxylamine to ammonium and of sulfite to sulfide. NrfA pentaheme nitrite reductase plays the key role in anaerobic nitrate respiration and the protection of bacterial cells from stresses caused by nitrogen oxides and hydrogen peroxide. Octaheme nitrite reductases from bacteria of the Thioalkalivibrio genus are less studied, and their function in the cell is unknown. In order to estimate the possible role of octaheme nitrite reductases in the cell resistance to oxidative stress, the peroxidase activity of the enzyme from T. nitratireducens (TvNiR) has been studied in detail. Comparative analysis of the active site structure of TvNiR and cytochrome c peroxidases has shown some common features, such as a five-coordinated catalytic heme and identical catalytic residues in active sites. A model of the possible productive binding of peroxide at the active site of TvNiR has been proposed. The peroxidase activity has been measured for TvNiR hexamers and trimers under different conditions (pH, buffers, the addition of CaCl2 and EDTA). The maximum peroxidase activity of TvNiR with ABTS as a substrate (k cat = 17 s­1; k cat/K m = 855 mM­1 s­1) has been 100­300 times lower than the activity of natural peroxidases. The different activities of TvNiR trimers and hexamers indicate that the rate-limiting stage of the reaction is not the catalytic event at the active site but the electron transfer along the heme c electron-transport chain.

Structural study of the X-ray-induced enzymatic reaction of octahaem cytochrome C nitrite reductase.

Octahaem cytochrome c nitrite reductase from the bacterium Thioalkalivibrio nitratireducens catalyzes the reduction of nitrite to ammonium and of sulfite to sulfide. The reducing properties of X-ray radiation and the high quality of the enzyme crystals allow study of the catalytic reaction of cytochrome c nitrite reductase directly in a crystal of the enzyme, with the reaction being induced by X-rays. Series of diffraction data sets with increasing absorbed dose were collected from crystals of the free form of the enzyme and its complexes with nitrite and sulfite. The corresponding structures revealed gradual changes associated with the reduction of the catalytic haems by X-rays. In the case of the nitrite complex the conversion of the nitrite ions bound in the active sites to NO species was observed, which is the beginning of the catalytic reaction. For the free form, an increase in the distance between the oxygen ligand bound to the catalytic haem and the iron ion of the haem took place. In the case of the sulfite complex no enzymatic reaction was detected, but there were changes in the arrangement of the active-site water molecules that were presumably associated with a change in the protonation state of the sulfite ions.

Structural and functional studies of multiheme cytochromes C involved in extracellular electron transport in bacterial dissimilatory metal reduction.

Bacteria utilizing insoluble mineral forms of metal oxides as electron acceptors in respiratory processes are widespread in the nature. The electron transfer from a pool of reduced quinones in the cytoplasmic membrane across the periplasm to the bacterial outer membrane and then to an extracellular acceptor is a key step in bacterial dissimilatory metal reduction. Multiheme cytochromes c play a crucial role in the extracellular electron transfer. The bacterium Shewanella oneidensis MR-1 was used as a model organism to study the mechanism of extracellular electron transport. In this review, we discuss recent data on the composition, structures, and functions of multiheme cytochromes c and their functional complexes responsible for extracellular electron transport in Shewanella oneidensis.

Nicotinamidase from the thermophilic archaeon Acidilobus saccharovorans: structural and functional characteristics.

Nicotinamidase is involved in the maintenance of NAD+ homeostasis and in the NAD+ salvage pathway of most prokaryotes, and it is considered as a possible drug target. The gene (ASAC_0847) encoding a hypothetical nicotinamidase has been found in the genome of the thermophilic archaeon Acidilobus saccharovorans. The product of this gene, NA_As0847, has been expressed in Escherichia coli, isolated, and characterized as a Fe(2+)-containing nicotinamidase (k(cat)/K(m) = 427 mM(-1)·sec(-1))/pyrazinamidase (k(cat)/K(m) = 331 mM(-1)·sec(-1)). NA_As0847 is a homodimer with molecular mass 46.4 kDa. The enzyme has high thermostability (T(1/2) (60°C) = 180 min, T(1/2) (80°C) = 35 min) and thermophilicity (T(opt) = 90°C, E(a) = 30.2 ± 1.0 kJ/mol) and broad pH interval of activity, with the optimum at pH 7.5. Special features of NA_As0847 are the presence of Fe2+ instead of Zn2+ in the active site of the enzyme and inhibition of the enzyme activity by Zn2+ at micromolar concentrations. Analysis of the amino acid sequence revealed a new motif of the metal-binding site (DXHXXXDXXEXXXWXXH) for homological archaeal nicotinamidases.

Identification of the ligand in the structure of the protein with unknown function STM4435 from Salmonella typhimurium.

The unidentified ligand, which is present in the crystal of the protein with unknown function STM4435 from Salmonella typhimurium, was identified using a combination of high-resolution X-ray crystallography and accurate-mass time-of-flight mass spectrometry. The identified glycerol was present as a component of the solutions used for the isolation and crystallization of the protein and serves as the ligand mimicking the natural metabolite, presumably, 2-keto-myo-isonitol, which is indicative of the involvement of STM4435 in the myo-isonitol catabolism. The results of the present study show that this approach holds promise in complex studies aimed at determining, refining, or confirming the protein functions.

Targeted delivery of siRNA to differentiated murine myotubes in culture by a conjugate of cationic oligopeptide with FS2 venom.

A conjugate of the ligand of FS2 venom dihydropyridine receptors with a cationic arginine-containing oligopeptide was synthesized. It was found that the conjugate provides siRNA delivery to murine myotubes differentiated in vitro. The effect of RNA interference with the use of siRNA complexes with the conjugate was observed when siRNA concentrations were an order of magnitude lower than those used in the case of siRNA complexes with a non-conjugated oligopeptide.

Identification of silver nanoparticles in the small intestinal mucosa, liver, and spleen of rats by transmission electron microscopy.

The effects of water-dispersed Ag nanoparticles on the small intestinal mucosa, liver, and spleen of rats were studied by transmission electron microscopy. Acute experiments demonstrated penetration of Ag nanoparticles injected into the isolated intestinal loop into the intestinal mucosa, liver, and splenic tissues. Ultrastructural changes (lobed nucleus, megamitochondria) were found in the studied organs. These data indicated that injection of water-dispersed Ag nanoparticles into the gastrointestinal tract was followed by their penetration through the epithelium of the small intestinal mucosa into other organs, e.g. into the liver and spleen. This fact is essential for evaluation of potential risks of the nanoparticle effects on human health and environment.

Influence of intermolecular contacts on the structure of recombinant prolidase from Thermococcus sibiricus.

Prolidases are peptidases that are specific for dipeptides with proline as the second residue. The structure of recombinant prolidase from the hyperthermophilic archaeon Thermococcus sibiricus (Tsprol) was determined at 2.6 Šresolution. The homodimer of Tsprol is characterized by a complete lack of interactions between the N- and C-terminal domains of the two subunits and hence can be considered to be the most open structure when compared with previously structurally studied prolidases. This structure exists owing to intermolecular coordination bonds between cadmium ions derived from the crystallization solution and histidine residues of a His tag and aspartate and glutamate residues, which link the dimers to each other. This linking leads to the formation of a crystal with a loose packing of protein molecules and low resistance to mechanical influence and temperature increase.

Expression, purification, crystallization and preliminary crystallographic analysis of a thermostable DNA ligase from the archaeon Thermococcus sibiricus.

DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl termini. Their function is essential to maintain the integrity of the genome in DNA replication, recombination and repair. A recombinant ATP-dependent DNA ligase from the hyperthermophilic anaerobic archaeon Thermococcus sibiricus was expressed in Escherichia coli and purified. Crystals were grown by vapour diffusion using the hanging-drop method with 17%(w/v) PEG 4000 and 8.5%(v/v) 2-propanol as precipitants. A diffraction experiment was performed with a single crystal, which diffracted X-rays to 3.0 Å resolution. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 58.590, b = 87.540, c = 126.300 Å.

ATP-dependent DNA ligase from Thermococcus sp. 1519 displays a new arrangement of the OB-fold domain.

DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl termini. Their function is essential for maintaining genome integrity in the replication, recombination and repair of DNA. High flexibility is important for the function of DNA ligase molecules. Two types of overall conformations of archaeal DNA ligase that depend on the relative position of the OB-fold domain have previously been revealed: closed and open extended conformations. The structure of ATP-dependent DNA ligase from Thermococcus sp. 1519 (LigTh1519) in the crystalline state determined at a resolution of 3.02 Šshows a new relative arrangement of the OB-fold domain which is intermediate between the positions of this domain in the closed and the open extended conformations of previously determined archaeal DNA ligases. However, small-angle X-ray scattering (SAXS) measurements indicate that in solution the LigTh1519 molecule adopts either an open extended conformation or both an intermediate and an open extended conformation with the open extended conformation being dominant.

Octaheme nitrite reductases: structure and properties.

Octaheme oxidoreductases are widespread among various bacterial taxa involved in the biogeochemical nitrogen cycle. The evolution of octaheme oxidoreductases of the nitrogen cycle from the evolutionarily more ancient pentaheme nitrite reductases was accompanied by changes in function from reduction of nitrogen oxides to their oxidation under changing environmental conditions. Octaheme nitrite reductases, which are the subject of the present review, are of a transitional form that combines structural and functional characteristics of pentaheme reductases and octaheme oxidases and possesses a number of unique features typical of only this family of enzymes. The review summarizes data on structure-function investigations of the family of octaheme nitrite reductases. Emphasis is given to comparison of the structures and functions of octaheme nitrite reductases and other multiheme oxidoreductases of the nitrogen cycle.

Iron-dependent superoxide dismutase from novel thermoacidophilic crenarchaeon Acidilobus saccharovorans: from gene to active enzyme.

A gene encoding superoxide dismutase was revealed in the genome of the thermoacidophilic crenarchaeon Acidilobus saccharovorans. A recombinant expression vector was constructed and transformed into E. coli cells. The novel recombinant superoxide dismutase was purified and characterized. The enzyme was shown to be an iron-dependent superoxide dismutase able to bind various bivalent metals in the active site. According to differential scanning calorimetric data, the denaturation temperature of the enzyme is 107.3°C. The maximal activity of the Fe(II) reconstituted enzyme defined by xanthine oxidase assay is 1700 U/mg protein. Study of the thermal stability of the superoxide dismutase samples with various metal contents by tryptophan fluorescence indicated that the thermal stability and activity of the enzyme directly depend on the nature of the reconstituted metal and the degree of saturation of binding sites.

[A new approach for study of structural and functional properties of proteins with unknown functions].

Selected proteins were produced in Escherichia coli bacterial expression system--three proteins from extremophil bacteria: a putative monooxygenase from Deinococcus radiodurans, a putative nucleotidyltransferase from Thermotoga maritima, a putative oxidoreductase from Exiguobacterium sibiricum; and a shaperon from Homo sapiens DJ-1. The protocol of isolation & purification of recombinant proteins were developed that allowed to obtain expression products with the purity of no less than 96%. Conditions for the crystallization have been selected that allowed a stable growth of crystals. Preliminary x-ray experiments were conducted in order to confirm the quality of produced crystals; the resolution of obtained structural data was from 1.2 to 1.8 angstrom.

[Analysis of functional properties of biologically active substances using eukaryotic cell models (review)].

This work presents an analysis of current data on the investigation into the functional properties of biologically active substances in model systems based on cultivated human cells. The knowledge regarding the practical application of cell cultures for the analysis of functional properties of bioactive substances is summarized, including antioxidant, immunomodulating, pro- and prebiotics, and chemoprevention properties. The most promising directions in cell culture model development for the investigation of functional properties, including three-dimensional models, are discussed.