Методом криоэлектронной микроскопии получена структура цитохром с нитритредуктазы из бактерии Thioalkalivibrio nitratireducens с разрешением 2.56 A. Проверена возможная структурная гетерогенность фермента, показано отсутствие значимых различий как в ходе полипептидной цепи, так и в ориентации боковых групп модели, полученной на основании данных криоэлектронной микроскопии, относительно структуры высокого разрешения, полученной методом рентгеноструктурного анализа.

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.

HU proteins are involved in bacterial DNA and RNA repair. Since these proteins are absent in cells of higher organisms, inhibitors of HU proteins can be used as effective and safe antibiotics. The crystallization conditions for the M. gallisepticum HU protein were found and optimized by the vapor-diffusion method. The X-ray diffraction data set was collected to 2.91 Å resolution from the crystals grown by the vapor-diffusion method on a synchrotron source. The crystals of the HU protein belong to sp. gr. P41212 and have the following unit-cell parameters: a = b = 97.94 Å, c = 77.92 Å, α = β = γ = 90°.

В НБИКС-Центре НИЦ “Курчатовский институт” в 2010 г. создано специальное подразделение – Белковая фабрика, которое во взаимодействии с Центром синхротронного излучения обеспечивает проведение работ в области структурной биологии. Рассмотрены технологические платформы, реализованные на базе Белковой фабрики, и возможности, которые она предоставляет исследователям. Кратко представлены основные проекты, которые реализуются в рамках Белковой фабрики в настоящее время.