The Role of Hyperhomocysteinemia in the Pathogenesis of Thromboocclusive Diseases

Homocysteine is a sulfur-containing non-proteinogenic amino acid that is an intermediate product of methionine metabolism. With excessive accumulation of homocysteine in the body, a state of hyperhomocysteinemia occurs, which has attracted the attention of doctors since the middle of the last century and received a serious impetus for research after the publication of data on the role of hyperhomocysteinemia in the pathogenesis of thromboocclusive diseases. To date, there are more than 7.5 thousand scientific papers devoted to the study of hyperhomocysteinemia, monothematic international conferences on clinical and therapeutic aspects of hypermonocysteinemia are held every year. In the Russian Federation, a detailed study of the role of homocysteine and its side effects in excess is of particular interest due to the prevalence of cardiovascular pathologies (according to statistics of the Ministry of Health of Russia-49.27% of deaths of Russians fall on this cohort of diseases). This is an important fact in Russian medicine, which deserves close attention from researchers and scientists.

Theanine Improves Salt Stress Tolerance via Modulating Redox Homeostasis in Tea Plants (Camellia sinensis L.)

Theanine, a unique non-proteinogenic amino acid, is one of the most abundant secondary metabolites in tea. Its content largely determines green tea quality and price. However, its physiological roles in tea plants remain largely unknown. Here, we showed that salt stress significantly increased the accumulation of glutamate, glutamine, alanine, proline, and γ-aminobutyric acid, as well as theanine, in the new shoots of tea plants. We further found that salt stress induced the expression of theanine biosynthetic genes, including CsGOGATs, CsAlaDC, and CsTSI, suggested that salt stress induced theanine biosynthesis. Importantly, applying theanine to the new shoots significantly enhanced the salt stress tolerance. Similar effects were also found in a model plant Arabidopsis. Notably, exogenous theanine application increased the antioxidant activity of the shoots under salt stress, suggested by reduced the reactive oxygen species accumulation and lipid peroxidation, as well as by the increased SOD, CAT, and APX activities and expression of the corresponding genes. Finally, genetic evidence supported that catalase-mediated antioxidant scavenging pathway is required for theanine-induced salt stress tolerance. Taken together, this study suggested that salt stress induces theanine biosynthesize in tea plants to enhance the salt stress tolerance through a CAT-dependent redox homeostasis pathway.

Improved pyrrolysine biosynthesis through phage assisted non-continuous directed evolution of the complete pathway

Pyrrolysine (Pyl, O) exists in nature as the 22nd proteinogenic amino acid. Despite being a fundamental building block of proteins, studies of Pyl have been hindered by the difficulty and inefficiency of both its chemical and biological syntheses. Here, we improve Pyl biosynthesis via rational engineering and directed evolution of the entire biosynthetic pathway. To accommodate toxicity of Pyl biosynthetic genes in Escherichia coli, we also develop Alternating Phage Assisted Non-Continuous Evolution (Alt-PANCE) that alternates mutagenic and selective phage growths. The evolved pathway provides 32-fold improved yield of Pyl-containing reporter protein compared to the rationally engineered ancestor. Evolved PylB mutants are present at up to 4.5-fold elevated levels inside cells, and show up to 2.2-fold increased protease resistance. This study demonstrates that Alt-PANCE provides a general approach for evolving proteins exhibiting toxic side effects, and further provides an improved pathway capable of producing substantially greater quantities of Pyl-proteins in E. coli.

GABA plays a key role in plant acclimation to a combination of high light and heat stress

ABSTRACTPlants are frequently subjected to different combinations of abiotic stresses, such as high light intensity and elevated temperatures. These environmental conditions pose an important threat to agriculture production, affecting photosynthesis and decreasing yield. Metabolic responses of plants, such as alterations in carbohydrates and amino acid fluxes, play a key role in the successful acclimation of plants to different abiotic stresses, directing resources towards stress responses and suppressing growth. Here we show that the primary metabolic response of Arabidopsis thaliana plants to high light or heat stress is different than that of plants subjected to a combination of high light and heat stress. We further demonstrate that a combination of high light and heat stress results in a unique metabolic response that includes increased accumulation of sugars and amino acids, coupled with decreased levels of metabolites participating in the tricarboxylic acid (TCA) cycle. Among the amino acids exclusively accumulated during a combination of high light and heat stress, we identified the non-proteinogenic amino acid γ-aminobutyric acid (GABA). Analysis of different mutants deficient in GABA biosynthesis, in particular two independent alleles of glutamate decarboxylase 3 (gad3), reveal that GABA plays a key role in the acclimation of plants to a combination of high light and heat stress. Taken together, our findings identify a new role for GABA in regulating plant responses to stress combination.One sentence summaryThe non-proteinogenic amino acid γ-aminobutyric acid (GABA) is required for plant acclimation to a combination of high light and heat stress in Arabidopsis.

Improved pyrrolysine biosynthesis through continuous directed evolution of the complete pathway

Pyrrolysine (Pyl, O) exists in nature as the 22nd proteinogenic amino acid. Despite being a fundamental building block of proteins, studies of Pyl have been hindered by the difficulty and inefficiency of both its chemical and biological syntheses. Here, we improved Pyl biosynthesis via rational engineering and directed evolution of the entire biosynthetic pathway. To accommodate toxicity of Pyl biosynthetic genes in Escherichia coli, we devised an approach termed Alternating Phage Assisted Non-Continuous Evolution (Alt-PANCE) that alternates mutagenic and selective phage growths. The evolved pathway exhibited a 32-fold improved yield of Pyl-containing super- folder green fluorescent protein (sfGFP) compared to the rationally engineered ancestor, whereas the WT pathway produced no detectable quantities of Pyl-containing sfGFP. This study demonstrates that Alt-PANCE provides a general approach for evolving proteins exhibiting toxic side effects, and further provides an improved pathway capable of producing substantially greater quantities of Pyl- proteins in E. coli.

Synthesis of l-Kynurenine and Homo-l-Kynurenine via an Aza-Fries Rearrangement

l-Kynurenine, a non-proteinogenic amino acid, is the primary metabolite of tryptophan via the kynurenine pathway. Kynurenine is involved in a variety of biological processes occurring in the human body, notably in the central nervous system. Thus, the study of this molecule offers multiple opportunities for drug discovery; however, an essential prelude for biological studies is to secure the supply of kynurenine and analogues thereof. A simple synthetic procedure for the efficient preparation of enantiomerically pure l-kynurenine from l-aspartic acid and its implementation to prepare homo-l-kynurenine from l-glutamic acid is presented. The approach relies on a photochemical aza-Fries rearrangement of the corresponding acyl-aniline as the fundamental transformation.