Vol 73, No 1 (2018)

Majority of native enzymes are poorly applicable for practical usage: that is why different methods of enzyme modification are used to obtain the biocatalysts with appropriate characteristics. Development of genome sequencing and various modern approaches in protein engineering allow one to identify protein of interest and to improve the enzyme properties for a particular process. This review describes the results on development of novel biocatalysts based on bioinformatics and rational design. New genes encoding formate dehydrogenase (FDH) from bacterium Staphylococcus aureus, yeasts Ogataea parapolymorpha and Saccharomyces cerevisiae and moss Physcomitrella patens (SauFDH, OpaFDH, SceFDH and PpaFDH, respectively), have been cloned. New FDHs were produced in the active form and characterized. SauFDH was shown to have at least 2-fold higher catalytic constant than other known FDHs. OpaFDH has catalytic parameters as good as those for soy FDH mutant forms, and in addition, is more thermostable. Apo- and holo-forms of SauFDH have been crystallized. Mutation of two Cys residues in Pseudomonas sp.101 enzyme (PseFDH) yields enzyme preparations with improved kinetic parameters and enhanced thermal and chemical stability. New generation of PseFDH preparations with the coenzyme specificity changed from NAD⁺ to NADP⁺ have been obtained. The effect of ionic liquids on the catalytic properties and thermal stability of six wild-type recombinant FDHs, and a number of their mutants, have been studied. In case of D-amino acid oxidase (DAAO), single-point mutations have been combined to create multi-point mutants. The introduced amino acid replacements have been shown to exert an additive effect, improving both kinetic parameters and increasing thermal and chemical stability. DAAO genes from Hansenula polymorpha yeast have been cloned. α-Amino acid ester hydrolase (AEH) gene has been cloned and expressed in the active form in E. coli. Structural modeling has been performed and the effectiveness in amino beta-lactams synthesis studied. The structure of a single-chain penicillin acylase from Alcaligenes faecalis (scAfPA) has been modeled and two variants of scAfPA gene was generated by PCR. Both variants have been expressed in E. coli, isolated and characterized. Catalytic properties of scAfPA were slightly better than those of its natural heterodimer.

Activation of antihypoxic program under the action of a number of transition and heavy metals has been studied using cell-based HIF1 ODD-luc and HRE-luc reporters. It has been demonstrated that Au³⁺, Pb²⁺, Sn²⁺, Hg²⁺ are weak HIF1 ODD-luc activators, likely reflecting their weak competition for the ironbinding site in the active center of HIF prolyl hydroxylase. Metals capable of replacing iron–Mn²⁺, Zn²⁺, Cu²⁺ и Ni²⁺–activate at high submillimolar concentrations, which indicates low permeability of the cell membrane for transition metals. The highest activation is observed for Co²⁺ and Cd²⁺, however, Cd²⁺ is highly toxic even at 10 μM, in contrast to Co²⁺, which activates both reporters without toxicity signs up to 25 μM for 24 h. A significant activation by Co²⁺ is observed already in low micromolar range of concentrations, which can be recommended for use in hypoxia mimicking.

The complex formation of TEM-1 β-lactamase and its three mutant forms TEM-32, TEM-37, and TEM-39 with substrates cephalothin and CENTA and serine beta-lactamase inhibitors sulbactam, tazobactam, and clavulanic acid is studied using the methods of molecular dynamics. It is found that the stability of the complexes is caused by the electrostatic attraction between the deprotonated carboxyl group of the β-lactam ring of the substrate (inhibitor) and the positively charged amino groups of the lysine 234 and 73 residues, located in the active site of the enzymes. The formation of a hydrogen bond between this substrate group or its carbonyl oxygen with the hydroxyl group of the catalytic serine 70 residue and also between the negatively charged substituent groups and the positive charge region formed by the arginine 244 guanidine group and the asparagine 276 amino group is observed for some complexes. The binding energy of CENTA with TEM-1 β-lactamase is below the analogous binding energy of cephalothin, which is confirmed by the values of the Michaelis constants, determined experimentally. It is also found that the inhibitors bind to the mutant forms of β-lactamases related to the inhibitor-resistant phenotype, with higher affinity than TEM-1 β-lactamase.

Analysis of the conformational variety of the oligosaccharide fragments of the human glycan receptors LSTa (α-D-Neu5Ac-(2-3)-β-D-Gal-(1-3)-β-D-GlcNAc-(1-3)-β-D-Gal(1-4)-D-Glc) and LSTc (α-D-Neu5Ac-(2-6)-β-D-Gal-(1-3)-β-D-GlcNAc-(1-3)-β-D-Gal(1-4)-D-Glc) in aqueous solution has been performed with the comprehensive use of molecular modeling and statistical data processing followed by determination of major and minor stabilized conformers and selection of relevant topologies. The sialic acid ring conformational free energy landscape for both pentasaccharides has been reconstructed and analyzed giving a specification of the most probable distorted ring conformations of the basic chair ¹C₄ structure. The obtained results are in a good agreement with experimental data generated by nuclear magnetic resonance spectroscopy and X-ray crystallography.