The Experts below are selected from a list of 16266 Experts worldwide ranked by ideXlab platform
Uwe T Bornscheuer - One of the best experts on this subject based on the ideXlab platform.
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synthesis of 1r 3r 1 amino 3 methylcyclohexane by an enzyme cascade reaction
Tetrahedron, 2016Co-Authors: Lilly Skalden, Christin Peters, Lukas Ratz, Uwe T BornscheuerAbstract:Abstract Amine Transaminases (ATAs) are powerful enzymes for the synthesis of chiral amines. Although the request for amines with more than one chiral center is increasing, their synthesis is still challenging. Here we show a casacde reaction combining an enoate reductase (ERED) and an amine transaminase (ATA-VibFlu), which allows access to optically pure (1R,3R)-1-amino-3-methylcyclohexane. Because all known wildtype EREDs show a (S)-selectivity for 3-methylcyclohex-2-enone and the ATA-VibFlu only showed a modest enantioselectivity, different variants of EREDs and ATAs were investigated and suitable mutant enzymes were identified. In whole cell biocatalyses using the ERED YqjM Cys26Asp/Ile69Thr and the ATA-VibFlu Leu56Ile (1R,3R)-1-amino-3-methylcyclohexane was obtained at high optical purity (97% de).
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Glycine Oxidase Based High-Throughput Solid-Phase Assay for Substrate Profiling and Directed Evolution of (R)- and (S)‑Selective Amine Transaminases
2015Co-Authors: Martin S. Weiß, Clare Vickers, Ioannis V Pavlidis, Matthias Höhne, Uwe T BornscheuerAbstract:Transaminases represent one of the most important enzymes of the biocatalytic toolbox for chiral amine synthesis as they allow asymmetric synthesis with quantitative yields and high enantioselectivity. In order to enable substrate profiling of Transaminases for acceptance of different amines, a glycine oxidase and horseradish peroxidase coupled assay was developed. Transaminase activity is detected upon transfer of an amine group from an amino donor substrate to glyoxylate, generating glycine, which is subsequently oxidized by glycine oxidase, releasing hydrogen peroxide in turn. Horseradish peroxidase uses the hydrogen peroxide to produce benzoquinone, which forms a red quinone imine dye by a subsequent condensation reaction. As glycine does not carry a chiral center, both (R)- and (S)-selective Transaminases accepting glyoxylate as amino acceptor are amenable to screening. The principle has been transferred to establish a high-throughput solid-phase assay which dramatically decreases the screening effort in directed evolution of Transaminases, as only active variants are selected for further analysis
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glycine oxidase based high throughput solid phase assay for substrate profiling and directed evolution of r and s selective amine Transaminases
Analytical Chemistry, 2014Co-Authors: Martin S Weis, Ioannis V Pavlidis, Clare Vickers, Matthias Hohne, Uwe T BornscheuerAbstract:Transaminases represent one of the most important enzymes of the biocatalytic toolbox for chiral amine synthesis as they allow asymmetric synthesis with quantitative yields and high enantioselectivity. In order to enable substrate profiling of Transaminases for acceptance of different amines, a glycine oxidase and horseradish peroxidase coupled assay was developed. Transaminase activity is detected upon transfer of an amine group from an amino donor substrate to glyoxylate, generating glycine, which is subsequently oxidized by glycine oxidase, releasing hydrogen peroxide in turn. Horseradish peroxidase uses the hydrogen peroxide to produce benzoquinone, which forms a red quinone imine dye by a subsequent condensation reaction. As glycine does not carry a chiral center, both (R)- and (S)-selective Transaminases accepting glyoxylate as amino acceptor are amenable to screening. The principle has been transferred to establish a high-throughput solid-phase assay which dramatically decreases the screening effor...
Helen C. Hailes - One of the best experts on this subject based on the ideXlab platform.
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a rapid sensitive colorimetric assay for the high throughput screening of Transaminases in liquid or solid phase
Chemical Communications, 2015Co-Authors: Damien Baud, John M. Ward, N Ladkau, Thomas S Moody, Helen C. HailesAbstract:A new colorimetric method has been developed to screen Transaminases using an inexpensive amine donor. The assay is sensitive, has a low level of background coloration, and can be used to identify and profile transaminase activities against aldehyde and ketone substrates in a high-throughput format. Significantly it is also amendable to solid phase colony screening.
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The substrate specificity, enantioselectivity and structure of the (R)-selective amine : pyruvate transaminase from Nectria haematococca
2014Co-Authors: Sayer C, Ruben J. Martinez-torres, Michail N. Isupov, Helen C. Hailes, Jennifer A. Littlechild, Richter N, Jm WardAbstract:During the last decade the use of Transaminases for the production of pharmaceutical and fine chemical intermediates has attracted a great deal of attention. Transaminases are versatile biocatalysts for the efficient production of amine intermediates and many have (S)-enantiospecificity. Transaminases with (R)-specificity are needed to expand the applications of these enzymes in biocatalysis. In this work we have identified a fungal putative (R)-specific transaminase from the Eurotiomycetes Nectria haematococca, cloned a synthetic version of this gene, demonstrated (R)-selective deamination of several substrates including (R)-a-methylbenzylamine, as well as production of (R)-amines, and determined its crystal structure. The crystal structures of the holoenzyme and the complex with an inhibitor gabaculine offer the first detailed insight into the structural basis for substrate specificity and enantioselectivity of the industrially important class of (R)-selective amine : pyruvate Transaminases
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The substrate specificity, enantioselectivity and structure of the (R)-selective amine : pyruvate transaminase from Nectria haematococca
FEBS Journal, 2014Co-Authors: C. Sayer, Ruben J. Martinez-torres, Nina Richter, Michail N. Isupov, Helen C. Hailes, Jennifer A. Littlechild, John M. WardAbstract:During the last decade the use of Transaminases for the production of pharmaceutical and fine chemical intermediates has attracted a great deal of attention. Transaminases are versatile biocatalysts for the efficient production of amine intermediates and many have (S)-enantiospecificity. Transaminases with (R)-specificity are needed to expand the applications of these enzymes in biocatalysis. In this work we have identified a fungal putative (R)-specific transaminase from the Eurotiomycetes Nectria haematococca, cloned a synthetic version of this gene, demonstrated (R)-selective deamination of several substrates including (R)-α-methylbenzylamine, as well as production of (R)-amines, and determined its crystal structure. The crystal structures of the holoenzyme and the complex with an inhibitor gabaculine offer the first detailed insight into the structural basis for substrate specificity and enantioselectivity of the industrially important class of (R)-selective amine : pyruvate Transaminases. Database The atomic coordinates and structure factors for the Nectria TAm in holoenzyme and gabaculine-bound forms have been deposited in the PDB as entries 4cmd and 4cmf respectively. Structured digital abstract • TAm and TAm bind by x-ray crystallography (View interaction)
John M. Ward - One of the best experts on this subject based on the ideXlab platform.
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a rapid sensitive colorimetric assay for the high throughput screening of Transaminases in liquid or solid phase
Chemical Communications, 2015Co-Authors: Damien Baud, John M. Ward, N Ladkau, Thomas S Moody, Helen C. HailesAbstract:A new colorimetric method has been developed to screen Transaminases using an inexpensive amine donor. The assay is sensitive, has a low level of background coloration, and can be used to identify and profile transaminase activities against aldehyde and ketone substrates in a high-throughput format. Significantly it is also amendable to solid phase colony screening.
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The substrate specificity, enantioselectivity and structure of the (R)-selective amine : pyruvate transaminase from Nectria haematococca
FEBS Journal, 2014Co-Authors: C. Sayer, Ruben J. Martinez-torres, Nina Richter, Michail N. Isupov, Helen C. Hailes, Jennifer A. Littlechild, John M. WardAbstract:During the last decade the use of Transaminases for the production of pharmaceutical and fine chemical intermediates has attracted a great deal of attention. Transaminases are versatile biocatalysts for the efficient production of amine intermediates and many have (S)-enantiospecificity. Transaminases with (R)-specificity are needed to expand the applications of these enzymes in biocatalysis. In this work we have identified a fungal putative (R)-specific transaminase from the Eurotiomycetes Nectria haematococca, cloned a synthetic version of this gene, demonstrated (R)-selective deamination of several substrates including (R)-α-methylbenzylamine, as well as production of (R)-amines, and determined its crystal structure. The crystal structures of the holoenzyme and the complex with an inhibitor gabaculine offer the first detailed insight into the structural basis for substrate specificity and enantioselectivity of the industrially important class of (R)-selective amine : pyruvate Transaminases. Database The atomic coordinates and structure factors for the Nectria TAm in holoenzyme and gabaculine-bound forms have been deposited in the PDB as entries 4cmd and 4cmf respectively. Structured digital abstract • TAm and TAm bind by x-ray crystallography (View interaction)
V Popov - One of the best experts on this subject based on the ideXlab platform.
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three dimensional structure of branched chain amino acid transaminase from thermoproteus uzoniensis in complex with l norvaline
Crystallography Reports, 2020Co-Authors: Konstantin M Boyko, V Popov, Yu A Nikolaeva, V I Timofeev, Yu E BezsudnovaAbstract:Transaminases (aminotransferases) are stereospecific enzymes catalyzing the reversible amino group transfer from various substrates. Transaminases are key enzymes in amino acid metabolism in all organisms, and they show promise for fine organic synthesis. Among a diversity of Transaminases, as-yet poorly characterized pyridoxal 5′-phosphate-dependent fold type IV Transaminases have attracted great interest. This transaminase family shows specificity for both D- and L-amino acids and (R)-amines. The crystal structure of thermally stable fold type IV branched-chain amino acid transaminase from the archaeon Thermoproteus uzoniensis in complex with the non-natural substrate L-norvaline was established. The mechanism of substrate binding is considered. The key amino acids involved in the substrate binding are described.
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diaminopelargonic acid transaminase from psychrobacter cryohalolentis is active towards s 1 phenylethylamine aldehydes and alpha diketones
Applied Microbiology and Biotechnology, 2018Co-Authors: Ekaterina Yu Bezsudnova, T N Stekhanova, A V Popinako, T V Rakitina, Alena Yu Nikolaeva, Konstantin M Boyko, V PopovAbstract:Substrate and reaction promiscuity is a remarkable property of some enzymes and facilitates the adaptation to new metabolic demands in the evolutionary process. Substrate promiscuity is also a basis for protein engineering for biocatalysis. However, molecular principles of enzyme promiscuity are not well understood. Even for the widely studied PLP-dependent Transaminases of class III, the reliable prediction of the biocatalytically important amine transaminase activity is still difficult if the desired activity is unrelated to the natural activity. Here, we show that 7,8-diaminopelargonic acid transaminase (synthase), previously considered to be highly specific, is able to convert (S)-(-)-1-phenylethylamine and a number of aldehydes and diketones. We were able to characterize the (S)-amine transaminase activity of 7,8-diaminopelargonic acid transaminase from Psychrobacter cryohalolentis (Pcryo361) and analyzed the three-dimensional structure of the enzyme. New substrate specificity for α-diketones was observed, though only a weak activity towards pyruvate was found. We examined the organization of the active site and binding modes of S-adenosyl-L-methionine and (S)-(-)-1-phenylethylamine using X-ray analysis and molecular docking. We suggest that the Pcryo361 affinity towards (S)-(-)-1-phenylethylamine arises from the recognition of the hydrophobic parts of the specific substrates, S-adenosyl-L-methionine and 7-keto-8-aminopelargonic acid, and from the flexibility of the active site. Our results support the observation that the conversion of amines is a promiscuous activity of many Transaminases of class III and is independent from their natural function. The analysis of amine transaminase activity from among various Transaminases will help to make the sequence-function prediction for biocatalysis more reliable.
Martin S Weis - One of the best experts on this subject based on the ideXlab platform.
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glycine oxidase based high throughput solid phase assay for substrate profiling and directed evolution of r and s selective amine Transaminases
Analytical Chemistry, 2014Co-Authors: Martin S Weis, Ioannis V Pavlidis, Clare Vickers, Matthias Hohne, Uwe T BornscheuerAbstract:Transaminases represent one of the most important enzymes of the biocatalytic toolbox for chiral amine synthesis as they allow asymmetric synthesis with quantitative yields and high enantioselectivity. In order to enable substrate profiling of Transaminases for acceptance of different amines, a glycine oxidase and horseradish peroxidase coupled assay was developed. Transaminase activity is detected upon transfer of an amine group from an amino donor substrate to glyoxylate, generating glycine, which is subsequently oxidized by glycine oxidase, releasing hydrogen peroxide in turn. Horseradish peroxidase uses the hydrogen peroxide to produce benzoquinone, which forms a red quinone imine dye by a subsequent condensation reaction. As glycine does not carry a chiral center, both (R)- and (S)-selective Transaminases accepting glyoxylate as amino acceptor are amenable to screening. The principle has been transferred to establish a high-throughput solid-phase assay which dramatically decreases the screening effor...