Exploring donor substrate promiscuity of a Thermostable Transketolase by directed evolution

Enzymes catalyzing asymmetric carboligation reactions typically show very high specificity for their nucleophilic substrate. Transketolase (TK, EC 2.2.1.1) catalyses a reversible transfer of a hydroxylated C2 fragment among phosphorylated ketoses and aldoses. [1] Native TK converts a large variety of (2R)-hydroxyaldehydes as the electrophilic acceptor substrates, but apart from its natural phosphoketose donors TK accepts only hydroxy­pyruvate (hydroxylated donor) (Figure 1). In contrast, 1-deoxy-D-xylulose-5-phosphate synthase (DXS, EC 2.2.1.7) catalyzes the specific decarboxylative transfer of the acetyl moiety from pyruvate (non-hydroxylated donor) to glyceraldehyde-3-phosphate to yield 1-deoxy-D-xylulose 5-phosphate (DXP), which constitutes the first step into the non-mevalonate biosynthesis of terpenoids (Figure 1).[2] Reactions of native TK and DXS are mutually exclusive in vivo. Please click Additional Files below to see the full abstract

Hydroxamate Assays for High‐Throughput Screening of Transketolase Libraries Against Arylated Substrates

We recently reported that the transketolase from Geobacillus stearothermophilus (TKgst) upon acyl transfer to nitrosoarenes generates N‐aryl hydroxamic acids (HA). The latter are metal chelating compounds that in the presence of Fe(III) ions form deep‐red complexes. Here, we applied this principle to the development of a colorimetric assay in both solid‐ and liquid‐phase formats for the high‐throughput screening of TKgst and its variants. Screening a set of positive hits from a L382X/D470X library validated the specificity and sensitivity of the assays. The solid surface assay allows a clear distinction between positive and negative colonies by the naked eye in qualitative mode, and further also to measure activity in semi‐quantitative fashion in the liquid‐phase format. The assay will be important for engineering the TKgst enzyme towards improved conversion of aromatic aldehydes by their close structural analogy to nitrosoarenes

Fluorogenic kinetic assay for high-throughput discovery of stereoselective ketoreductases relevant to pharmaceutical synthesis

Enantiomerically pure 1-(6-methoxynaphth-2-yl) and 1-(6-(dimethylamino)naphth-2-yl) carbinols are fluorogenic substrates for aldo/keto reductase (KRED) enzymes, which allow the highly sensitive and reliable determination of activity and kinetic constants of known and unknown enzymes, as well as an immediate enantioselectivity typing. Because of its simplicity in microtiter plate format, the assay qualifies for the discovery of novel KREDs of yet unknown specificity among this vast enzyme superfamily. The suitability of this approach for enzyme typing is illustrated by an exemplary screening of a large collection of short-chain dehydrogenase/reductase (SDR) enzymes arrayed from a metagenomic approach. We believe that this assay format should match well the pharmaceutical industry’s demand for acetophenone-type substrates and the continuing interest in new enzymes with broad substrate promiscuity for the synthesis of chiral, non-racemic carbinols

An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution

Defined sialoglycoconjugates are important molecular probes for studying the role of sialylated glycans in biological systems. We show that the α2,3‐sialyltransferase from Photobacterium phosphoreum JT‐ISH‐467 (2,3SiaTpph) tolerates a very broad substrate scope for modifications in the sialic acid part, including bulky amide variation, C5/C9 substitution, and C5 stereoinversion. To reduce the enzyme's hydrolytic activity, which erodes the product yield, an extensive structure‐guided mutagenesis study identified three variants that show up to five times higher catalytic efficiency for sialyltransfer, up to ten times lower efficiency for substrate hydrolysis, and drastically reduced product hydrolysis. Variant 2,3SiaTpph (A151D) displayed the best performance overall in the synthesis of the GM3 trisaccharide (α2,3‐Neu5Ac‐Lac) from lactose in a one‐pot, two‐enzyme cascade. Our study demonstrates that several complementary solutions can be found to suppress the common problem of undesired hydrolysis activity of microbial GT80 sialyltransferases. The new enzymes are powerful catalysts for the synthesis of a wide variety of complex natural and new‐to‐nature sialoconjugates for biological studies

Особенности оценки профессиональных рисков в медицинской организации

Работа посвящена оценки профессионального риска для работников медицинских организаций с учетом эпидемиологической обстановки. В данной работе рассмотрено нормативно-правовое регулирование оценки профессионального риска в Российской Федерации. Проведен анализ существующих методов оценки профессионального риска. В результате исследования проведена оценка профессионального риска для работников медицинской организации и предложены мероприятия по уменьшению уровня риска.The work is devoted to the assessment of professional risk for employees of medical organizations, taking into account the epidemiological situation. This paper examines the legal regulation of professional risk assessment in the Russian Federation. The analysis of existing methods of professional risk assessment is carried out. As a result of the study, an assessment of professional risk for employees of a medical organization was carried out and measures to reduce the level of risk were proposed

Combining aldolases and transaminases for the synthesis of 2‑amino-4-hydroxybutanoic acid

Amino acids are of paramount importance as chiral building blocks of life, for drug development in modern medicinal chemistry, and for the manufacture of industrial products. In this work, the stereoselective synthesis of (S)- and (R)-2-amino-4-hydroxybutanoic acid was accomplished using a systems biocatalysis approach comprising a biocatalytic one-pot cyclic cascade by coupling of an aldol reaction with an ensuing stereoselective transamination. A class II pyruvate aldolase from E. coli, expressed as a soluble fusion protein, in tandem with either an S- or R-selective, pyridoxal phosphate dependent transaminase was used as a catalyst to realize the conversion, with formaldehyde and alanine being the sole starting materials. Interestingly, the class II pyruvate aldolase was found to tolerate formaldehyde concentrations of up to 1.4 M. The cascade system was found to reach product concentrations for (S)- or (R)-2-amino-4-hydroxybutanoic acid of at least 0.4 M, rendering yields between 86% and >95%, respectively, productivities of >80 g L–1 d–1, and ee values of >99%.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 635595 (CarbaZymes), the Ministerio de Economía y Competitividad (MINECO), the Fondo Europeo de Desarrollo Regional (FEDER) (grant no. CTQ2015-63563-R to P.C.), and COST action CM1303 Systems Biocatalysis.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Исследование последовательности импульсов тормозного излучения малогабаритного бетатрона

Разработана имитационная модель потока импульсного тормозного излучения и реализована в подпрограмме на MathCad. Адаптирована модель потока импульсного тормозного излучения к имитационной модели формирования цифровых радиографических изображений. Исследовано влияние параметров исходного потока тормозного излучения на качество цифровых радиографических изображений. Проведён цикл натурных экспериментов по оценке изменения параметров потока импульсов регистрируемого излучения с цифровых детекторов на этапах формирования радиографического изображения на комплексе высокоэнергетической цифровой радиографии Томского политехнического университета.A simulation model of pulsed radiation flux and implemented in a subroutine on MathCad is available. Pulse bremsstrahlung flow model for a simulation model of digital radiographic image formation Adapted. Effect of source stream parameters. Experiments on the study of changes in the parameters of the pulse flux are recorded using digital detectors at the stages of radiographic image formation on the complexes of high-energy digital radiography of the Tomsk Polytechnic University

Enantioselective Synthesis of Pharmaceutically Relevant Bulky Arylbutylamines Using Engineered Transaminases

ATAs engineered for having an enlarged small binding pocket were applied for the synthesis of enantiomerically pure (R)‐benzo[1,3]dioxol‐5‐yl‐butylamine, a chiral component of human leukocyte elastase inhibitor DMP 777 (L‐694,458). Kinetic resolution of the racemic amine was performed by using the L59A variant of the (S)‐selective ATA from Chromobacterium violaceum (Cv‐ATA), providing the residual (R)‐enantiomer in excellent yield and >99% ee. At moderate enzyme loading and absence of co‐solvent, high volumetric productivity of 0.22 mol L⁻¹ h⁻¹ (42.5 g L⁻¹ h⁻¹) was achieved. Complementarily, the (S)‐enantiomer was generated via kinetic resolution using the (R)‐selective ATA‐117‐Rd11 from Arthrobacter sp. with acetone as the amino acceptor. In an alternative approach, we employed ATA‐117‐Rd11 for the asymmetric amination of the prochiral ketone precursor, which at 86% conversion gave the (R)‐benzo[1,3]dioxol‐5‐yl‐butylamine with excellent >99% ee. We further evaluated the utility of Cv‐ATA L59A for the asymmetric synthesis of pharmaceutically relevant (S)‐1‐phenylbutan‐1‐amine, a chiral component of the deubiquitinase inhibitor degrasyn (WP1130). The enzyme showed good tolerance to high concentrations of isopropylamine, producing (S)‐1‐phenylbutan‐1‐amine in enantiomerically pure form (>99% ee)

Biocatalytic Aldol Addition of Simple Aliphatic Nucleophiles to Hydroxyaldehydes

This ACS article is provided to You under the terms of this Standard ACS AuthorChoice/Editors' Choice usage agreement between You and the American Chemical Society ("ACS")(https://pubs.acs.org/page/policy/authorchoice_termsofuse.html)Asymmetric aldol addition of simple aldehydes and ketones to electrophiles is a cornerstone reaction for the synthesis of unusual sugars and chiral building blocks. We investigated -fructose-6-phosphate aldolase from E. coli (FSA) D6X variants as catalysts for the aldol additions of ethanal and nonfunctionalized linear and cyclic aliphatic ketones as nucleophiles to nonphosphorylated hydroxyaldehydes. Thus, addition of propanone, cyclobutanone, cyclopentanone, or ethanal to 3-hydroxypropanal or (S)- or (R)-3-hydroxybutanal catalyzed by FSA D6H and D6Q variants furnished rare deoxysugars in 8-77% isolated yields with high stereoselectivity (97:3 dr and >95% ee)