Crystals of the enzyme purine nucleoside phosphorylase from the extremophilic bacterium Halomonas Chromatireducens AGD 8-3, suitable for X-ray diffraction, were grown by the vapor-diffusion method. The X-ray diffraction data were collected from these crystals at the Belok beamline of the Kurchatov synchrotron radiation source (National Research Centre “Kurchatov Institute”) at 100 K to 1.80 Å resolution. The X-ray diffraction data were processed in the space groups P 1, P 2, P 2 1 , and P 622. The structure was solved by the molecular replacement method taking into account the twinning in the space groups P 2 1 and P 1 with one and two hexamers of the enzyme per asymmetric unit, respectively.

Mutant uridine phosphorylase genes from Shewanella oneidensis MR-1 ( S. oneidensis ) were constructed by site-directed mutagenesis and strains-producers of the corresponding recombinant (F5I and F5G) proteins were obtained on the basis of Escherichia coli cells. The mutant proteins were purified and their physicochemical and enzymatic properties were studied. It was shown that the N-terminal fragment of uridine phosphorylase plays an important role in the thermal stabilization of the enzyme as a whole. The role of the aminoacid (a.a.) residue phenylalanine (F5) in the formation of thermotolerance of uridine phosphorylases from gamma-proteobacteria was revealed.

Crystals of mutants of uridine phosphorylase from Shewanella oneidensis MR-1 at the active-site threonine residue were obtained, and the three-dimensional structures of the mutants were determined. It was shown that the loop 161–179 responsible for the nucleoside recognition is involved in stabilization of the hexameric structure of the protein and its disorder significantly facilitates the access of nucleoside to the enzyme active site. The role of conformational changes in the enzyme function is discussed.

Thymidine phosphorylase (deoA) and purine nucleoside phosphorylase (deoD) genes from the extremophilic bacterium Halomonas chromatireducens AGD 8-3 have been cloned. Expression plasmids were constructed, and highly efficient recombinant producer strains were obtained for these proteins. Recombinant nucleoside phosphorylases were isolated by ion-exchange chromatography in a homogeneous state, and their physical and enzymatic properties were studied. It was shown that the studied thymidine phosphorylase (HrTPP) and purine nucleoside phosphorylase (HrPNP) form the dimeric and hexameric forms, respectively. It was shown that the specific activity of HrTPP from the extremophilic bacterium H. chromatireducens AGD 8-3 was higher relative to thymidine (in comparison with its counterpart from E. coli).

A library of strains producing recombinant nucleoside phosphorylases (NPs) and their mutant and hybrid forms from various mesophilic and extremophilic microorganisms was constructed based on Escherichia coli cells. Substrates were shown to stabilize the NP structure upon thermal exposure, with the inorganic phosphate ion playing a decisive role in the process. Bioinformatics analyses made it possible to assume that the N‑terminal structure of NPs is largely responsible for their thermal stability. A hybrid thymidine phosphorylase (TPP) was constructed via the replacement of the N-terminal fragment (amino acid residues 1–62) of E. coli TPP with the corresponding TPP fragment from the thermophilic bacterium Geobacillus stearothermophilus. Higher thermal stability was observed for the hybrid TPP. The primary structure of E. coli uridine phosphorylase (UDP) was found to have a sequence, 25-Pro-Gly-Asp-Pro-30 (amino acid residues are numbered as in E. coli UDP), that is highly conserved among UDPs of mesophilic microorganisms. The E. coli UDP (Asp27Gly) mutant was constructed and similarly showed a higher thermal stability than the original form. The architecture of the phosphate-binding site and features of its function were assumed to be crucial for the thermal stability of the enzyme.

The characteristics of recombinant phytase from Obesumbacterium proteus have been studied after its integration into Yarrowia lipolytica with a Po1f (pUV3-Op) plasmid. Phytase activity was observed in Y. lipolytica Po1f transformants in a wide pH range (3.5–7.5) and remained at 80% of the initial value after 3-min heating at 95°C, which made it possible to add the enzyme to a fodder produced with heat treatment. In the case of transformant cultivation on low-grade vegetable substrates, they also possessed confirmed phytase activity, as well as an increase in the content of intracellular phosphates as compared to cells of the wild-type Y. lipolytica strain.

For the detection of sheep milk XO activity a various natural and artificial antioxidants were examined. Among the natural antioxidants L-cysteine was more effective in the stabilization of XO in heated milk XO of sheep milk activated by heat treatment in the presence of cysteine and molybdenum became able to convert nitrate and nitrite to nitric oxide (NO). Therefore, L-cysteine was used for double purposes: as the protector of enzyme active center against the oxidation during heat treatment of milk and as a reagent for S-nitrosothiol formation. Hypoxanthine, a natural substrate of XO, was the effective electron donor for NaR and NiR activities. Heat treatment of the milk in the presence of exogenous lecithin increased the activity of NaR and NiR of XO and CysNO formation. Thus, during the heat treatment: a) excess of exogenous phospholipids disintegrate the structure of MFGM and b) enzyme molecules denatured partially and their active center became available for exogenous cysteine, molybdenum, hypoxanthine and nitrate or nitrite.

Series of mutant genes of prokaryotic uridine phosphorylases (Shewanella oneidensis MR-1, Escherichia coli) were constructed, and the resulting strains-producers of the corresponding proteins were obtained. Proteins were purified, and their physicochemical and fermentative properties were studied. On the basis of the obtained data, the role of individual amino acid residues of the polypeptide chain of uridine phosphorylases in the formation and functioning of the phosphate-binding site in these proteins was shown. The assumption of independent binding of two substrates, ion of inorganic phosphate (Pi) and uridine (Urd), by nucleoside phosphorylases, was made.

A new artificial gene encoding human ω-amidase (Nit2) adapted for highly efficient expression in E. coli has been established. A pQE-Nit2 plasmid construct controlled by the T5 promoter has been engineered for its expression. The nit2 gene within the pQE-Nit2 construct has optimized codon usage and an artificial 6His-tag sequence inserted directly after the ATG initiation codon. This tag provides the possibility of single-step purification of a product via metal chelate chromatography. The codon-usage optimization involves the inclusion of several codons of extremely rare occurrence in natural E. coli ORFs within a 30 a.a-long N-terminal region. Other codons included in the N-terminus have moderate occurrence in E. coli. The subsequent sequence of the artificial gene has been composed of the most frequently occurring codons in E. coli. The recombinant producer based on the pQE-Nit2 construct allowed purification of the enzyme with an activity of 6.2 ± 0.2 μmol/min/mg protein, which corresponds to or slightly exceeds the specific activity of rat liver Nit2. The omega-amidase preparation is necessary for the screening of potential inhibitors that can be used as candidate drugs to cure hyperammonemia disorders in liver pathologies and oncological diseases.

Growth in the presence of vanadate and dissimilatory vanadate reduction under alkaline conditions were shown for a number of haloalkaliphilic Halomonas strains. Vanadate, which contains five-valent vanadium, was reduced to four- or three-valent compounds. Nitrate reductase plays the key role in vanadate reduction under alkaline conditions. The compounds containing reduced vanadium were obtained in crystalline form.

In this study we investigated the activities of antioxidant enzymes (superoxide dismutases (SODs) and catalases (CATs)) and the ROS level in cells of Yarrowia lipolytica yeasts grown in a medium with different pH values (4.5, 5.5 and 9.0). It was shown that an increase in the cellular ROS level took place under both acid and alkaline conditions. The growth under extreme conditions was accompanied by a significant increase of SOD activity (by 2.5 times in the acid medium and by 4 times in the alkaline medium), but catalase activity did not change. A study of the electrophoretic profile of catalases showed the presence of three isoforms differing in inhibitor resistance. The electrophoretic profiles of SODs and their inhibitory analysis revealed there are two other isoforms, probably of mitochondrial origin, in addition to Cu and Zn SOD. The role of SOD in pH-adaptation of extremophilic Y lipolytica yeasts is discussed.