Crystals of tryptophan indole-lyase and tyrosine phenol-lyase form stable quinonoid complexes.

The binding of substrates and inhibitors to wild-type Proteus vulgaris tryptophan indole-lyase and to wild type and Y71F Citrobacter freundii tyrosine phenol-lyase was investigated in the crystalline state by polarized absorption microspectrophotometry. Oxindolyl-lalanine binds to tryptophan indole-lyase crystals to accumulate predominantly a stable quinonoid intermediate absorbing at 502 nm with a dissociation constant of 35 microm, approximately 10-fold higher than that in solution. l-Trp or l-Ser react with tryptophan indole-lyase crystals to give, as in solution, a mixture of external aldimine and quinonoid intermediates and gem-diamine and external aldimine intermediates, respectively. Different from previous solution studies (Phillips, R. S., Sundararju, B.,Faleev, N. G. (2000) J. Am. Chem. Soc. 122, 1008-1114), the reaction of benzimidazole and l-Trp or l-Ser with tryptophan indole-lyase crystals does not result in the formation of an alpha-aminoacrylate intermediate, suggesting that the crystal lattice might prevent a ligand-induced conformational change associated with this catalytic step. Wild-type tyrosine phenol-lyase crystals bind l-Met and l-Phe to form mixtures of external aldimine and quinonoid intermediates as in solution. A stable quinonoid intermediate with lambda(max) at 502 nm is accumulated in the reaction of crystals of Y71F tyrosine phenol-lyase, an inactive mutant, with 3-F-l-Tyr with a dissociation constant of 1 mm, approximately 10-fold higher than that in solution. The stability exhibited by the quinonoid intermediates formed both by wild-type tryptophan indole-lyase and by wild type and Y71F tyrosine phenol-lyase crystals demonstrates that they are suitable for structural determination by x-ray crystallography, thus allowing the elucidation of a key species of pyridoxal 5'-phosphate-dependent enzyme catalysis

Innovations of the new public administration language: professionally prestigious sociolectisms or the signs of communicative failures?

This article is devoted to the study of the formation and functioning of professional innovations in the language of Russian public administration, reflecting the manifestation of tendencies toward standardization and informality in the choice of lexical items and syntactic constructions. To conduct a research, we used methods of linguistic observation and description, methods of component, comparative and lexicographic analysis, as well as the methodology of complex cognitively oriented synchronous diachronic analysis of language subsystems and their units. Printed text versions of speeches by people in the Government and the Federal Assembly of the Russian Federation were the materials for the study. A language analysis of the new public administration made it possible to single out a whole series of lexical-semantic, word-formative, morphological and syntactic innovations, which are identification marks of the studied subsystem and necessary elements of the thesaurus of a civil servant personality. A special group of language innovations of the new public administration is formed by the so-called thematically reoriented semantic neologisms. It is established that the language development of the new public administration is on the way to expand the field of functioning and strengthening its influence on the linguistic taste formation of Russian society. It is noted that the written form of communication in the field of public administration is significantly influenced by the oral colloquial element, and the results of this effect are manifested in all aspects: from lexical to communicative-pragmatic. Researchers are particularly worried about the consolidation in the professional language of the studied sphere of linguistic items violating linguistic and ethical-moral standards that create barriers for effective communication. In conclusion, it is unacceptable to actualize reduced professional language elements that contribute to the inefficiency of communication between representatives of authority and the community

Crystal Structure of Citrobacter freundii Asp214Ala Tyrosine Phenollyase Reveals that Asp214 is Critical for Maintaining a Strain in the Internal Aldimine

Tyrosine phenol-lyase (TPL) is a pyridoxal-5′-phosphate (PLP) dependent enzyme which catalyzes β-elimination of L-tyrosine. In the holoenzyme the protonated pyridinium N1 atom of the PLP cofactor is hydrogen-bonded to the side chain of Asp214. Here we report the X-ray structure of C. freundii D214A TPL determined at 1.9 Å resolution. Comparison with the structure of the wild-type TPL shows that the D214A replacement induced significant conformational reorganization in the active site leading to its partial closure. Significantly, in D214A TPL the strain in the internal aldimine is completely released and the pyridine N1 atom of PLP is deprotonated. These observations explain the considerably reduced activity of the D214A TPL towards its substrates [T. V. Demidkina et al., Biochim. Biophys. Acta, Proteins Proteomics 1764 (2006) 1268–1276]. The reported structure reveals that Asp214 is critical for maintaining the strain in the internal aldimine. We argue that this strain is used by the enzyme to accelerate the transaldimination reaction, the first step in the enzymatic catalysis.(doi: 10.5562/cca1915

engineering methionine γ lyase from citrobacter freundii for anticancer activity

Abstract Methionine deprivation of cancer cells, which are deficient in methionine biosynthesis, has been envisioned as a therapeutic strategy to reduce cancer cell viability. Methionine γ-lyase (MGL), an enzyme that degrades methionine, has been exploited to selectively remove the amino acid from cancer cell environment. In order to increase MGL catalytic activity, we performed sequence and structure conservation analysis of MGLs from various microorganisms. Whereas most of the residues in the active site and at the dimer interface were found to be conserved, residues located in the C-terminal flexible loop, forming a wall of the active site entry channel, were found to be variable. Therefore, we carried out site-saturation mutagenesis at four independent positions of the C-terminal flexible loop, P357, V358, P360 and A366 of MGL from Citrobacter freundii, generating libraries that were screened for activity. Among the active variants, V358Y exhibits a 1.9-fold increase in the catalytic rate and a 3-fold increase in KM, resulting in a catalytic efficiency similar to wild type MGL. V358Y cytotoxic activity was assessed towards a panel of cancer and nonmalignant cell lines and found to exhibit IC50 lower than the wild type. The comparison of the 3D-structure of V358Y MGL with other MGL available structures indicates that the C-terminal loop is either in an open or closed conformation that does not depend on the amino acid at position 358. Nevertheless, mutations at this position allosterically affects catalysis

THE EAEU IN A CHANGING GEOPOLITICAL CONTEXT: PRIORITIES FOR INTERNATIONAL COOPERATION

The article focuses on the international activity of the Eurasian Economic Union (EAEU) as an actor in the global economy with a full-fledged legal standing. It analyzes the current state of Eurasian integration within international cooperation and spotlights its correlations with intergovernmental organizations and integration associations. By means of examining the Eurasian Economic Commission’s (EEC’s) regulatory documents, the Main Directions of the EAEU International Activities for 2019 and the agenda of the Russian EAEU presidency in 2018, the author determines key conceptual approaches to evaluation and prognosis of the EAEU integration strategy. By applying comparative analysis, the author studies the experience of other integration associations relevant to the EAEU. The article determines the highestpotential models of EAEU international cooperation with due regard to the unstable global economic and geopolitical context. The author points out priority directions of the EAEU cooperation with an eye on strategic interests of the Russian Federation, differentiating them according to geographic areas of focus and taking into account the current state, risks and opportunities. The author concludes the article by giving recommendations on the implementation of the international agenda of the EAEU on a long-, midand short-term horizon. The author offers her ideas on further development of the EAEU international cooperation with a view to maximizing economic benefits for both Russia and the Union with future partnership in mind