Transketolase from Geobacillus stearothermophilus : characterization and modification of its enantioselectivity by protein engineering

La transcétolase (TK, EC 2.2.1.1) est une enzyme catalysant la formation de cétoses de configuration D-thréo à partir d’aldéhydes α-hydroxylés (2R), par formation stéréospécifique d’une liaison C-C. L’objectif de ces travaux est d’inverser l’énantiosélectivité de cette enzyme par ingénierie afin d’obtenir des cétoses L-érytho (recherchés pour leurs applications potentielles dans les domaines pharmaceutique et/ou nutritionnel) à partir d’aldéhydes α-hydroxylés (2S). Dans ce but, une TK thermostable (mTKgst) issue de la bactérie thermophile Geobacillus stearothermophillus a d’abord été identifiée et produite. L’étude de sa structure tridimensionnelle a permis d’identifier deux résidus du site actif ayant un rôle potentiel dans l’inversion de son énantiosélectivité : Leu382 et Asp470. Des banques demTKgst mutées ont alors été créées, selon deux stratégies : rationnelle et semi-rationnelle. La première a consisté à muter les deux résidus sélectionnés par mutagenèse par saturation de site, tandis que la seconde a consisté à modifier deux séquences de cinq résidus contigus à aux positions clés, selon la mutagenèse par cassette. Afin d’identifier les mTKgst mutées d’intérêt, un test de criblage à haut-débit a été mis au point, basé sur le suivi pH-métrique de la réaction en présence de rouge de phénol. A l’issue du criblage, le variant mTKgst-L382D/D470S a été mis en évidence. Son activité vis-à-vis d’un aldéhyde modèle de configuration (2S) a été augmentée d’un facteur 5 par rapport à l’enzyme sauvage et la perte de l’énantiosélectivité vis-à-vis desaldéhydes (2R) a été confirmée.Transketolase (TK, EC 2.2.1.1) catalyzes the formation of D-threo ketoses from (2R)-α-hydroxyaldehydes by the stereospecific formation of a C-C bond. Our aim was to invert the enantioselectivity of TK by protein engineering in order to obtain L-erytho ketoses (sought after for their potential pharmaceutical and/or nutritional applications) from (2S)-α-hydroxyaldehydes. For that purpose, a thermostable TK from thermophilic bacterium Geobacillus stearothermophilus (mTKgst) has been identified and overexpressed. After the study of the 3D-structure of mTKgst, two residues located in its active site (Leu382 and Asp470) were selected as mutation targets for the inversion of the enzyme’s enantioselectivity. Both rational and semi-rational approaches were considered for the construction of the mutant mTKgst libraries. In the former, the two residues were modified by site-saturation mutagenesis. In the latter, short sequences of five amino acids, neighboring target ones, were modified using a cassette mutagenesis technique. A novel continuous pH-based assay has been developed for the high-throughput screening of the mTKgst libraries, using phenol red as pH indicator. The screening revealed mTKgst-L382D/D470S as the top mutant, showing a 5-fold activity improvement towards a model (2S)-hydroxyaldehyde and the loss of enantioselectivity towards the (2R)-aldehyde

Etude de la transcétolase de Geobacillus stearothermophilus et modification de son énantiosélectivité par ingénierie enzymatique

Transketolase (TK, EC 2.2.1.1) catalyzes the formation of D-threo ketoses from (2R)-α-hydroxyaldehydes by the stereospecific formation of a C-C bond. Our aim was to invert the enantioselectivity of TK by protein engineering in order to obtain L-erytho ketoses (sought after for their potential pharmaceutical and/or nutritional applications) from (2S)-α-hydroxyaldehydes. For that purpose, a thermostable TK from thermophilic bacterium Geobacillus stearothermophilus (mTKgst) has been identified and overexpressed. After the study of the 3D-structure of mTKgst, two residues located in its active site (Leu382 and Asp470) were selected as mutation targets for the inversion of the enzyme’s enantioselectivity. Both rational and semi-rational approaches were considered for the construction of the mutant mTKgst libraries. In the former, the two residues were modified by site-saturation mutagenesis. In the latter, short sequences of five amino acids, neighboring target ones, were modified using a cassette mutagenesis technique. A novel continuous pH-based assay has been developed for the high-throughput screening of the mTKgst libraries, using phenol red as pH indicator. The screening revealed mTKgst-L382D/D470S as the top mutant, showing a 5-fold activity improvement towards a model (2S)-hydroxyaldehyde and the loss of enantioselectivity towards the (2R)-aldehyde.La transcétolase (TK, EC 2.2.1.1) est une enzyme catalysant la formation de cétoses de configuration D-thréo à partir d’aldéhydes α-hydroxylés (2R), par formation stéréospécifique d’une liaison C-C. L’objectif de ces travaux est d’inverser l’énantiosélectivité de cette enzyme par ingénierie afin d’obtenir des cétoses L-érytho (recherchés pour leurs applications potentielles dans les domaines pharmaceutique et/ou nutritionnel) à partir d’aldéhydes α-hydroxylés (2S). Dans ce but, une TK thermostable (mTKgst) issue de la bactérie thermophile Geobacillus stearothermophillus a d’abord été identifiée et produite. L’étude de sa structure tridimensionnelle a permis d’identifier deux résidus du site actif ayant un rôle potentiel dans l’inversion de son énantiosélectivité : Leu382 et Asp470. Des banques demTKgst mutées ont alors été créées, selon deux stratégies : rationnelle et semi-rationnelle. La première a consisté à muter les deux résidus sélectionnés par mutagenèse par saturation de site, tandis que la seconde a consisté à modifier deux séquences de cinq résidus contigus à aux positions clés, selon la mutagenèse par cassette. Afin d’identifier les mTKgst mutées d’intérêt, un test de criblage à haut-débit a été mis au point, basé sur le suivi pH-métrique de la réaction en présence de rouge de phénol. A l’issue du criblage, le variant mTKgst-L382D/D470S a été mis en évidence. Son activité vis-à-vis d’un aldéhyde modèle de configuration (2S) a été augmentée d’un facteur 5 par rapport à l’enzyme sauvage et la perte de l’énantiosélectivité vis-à-vis desaldéhydes (2R) a été confirmée

Etude de la transcétolase de Geobacillus stearothermophilus et modification de son énantiosélectivité par ingénierie enzymatique

La transcétolase (TK, EC 2.2.1.1) est une enzyme catalysant la formation de cétoses de configuration D-thréo à partir d aldéhydes a-hydroxylés (2R), par formation stéréospécifique d une liaison C-C. L objectif de ces travaux est d inverser l énantiosélectivité de cette enzyme par ingénierie afin d obtenir des cétoses L-érytho (recherchés pour leurs applications potentielles dans les domaines pharmaceutique et/ou nutritionnel) à partir d aldéhydes a-hydroxylés (2S). Dans ce but, une TK thermostable (mTKgst) issue de la bactérie thermophile Geobacillus stearothermophillus a d abord été identifiée et produite. L étude de sa structure tridimensionnelle a permis d identifier deux résidus du site actif ayant un rôle potentiel dans l inversion de son énantiosélectivité : Leu382 et Asp470. Des banques demTKgst mutées ont alors été créées, selon deux stratégies : rationnelle et semi-rationnelle. La première a consisté à muter les deux résidus sélectionnés par mutagenèse par saturation de site, tandis que la seconde a consisté à modifier deux séquences de cinq résidus contigus à aux positions clés, selon la mutagenèse par cassette. Afin d identifier les mTKgst mutées d intérêt, un test de criblage à haut-débit a été mis au point, basé sur le suivi pH-métrique de la réaction en présence de rouge de phénol. A l issue du criblage, le variant mTKgst-L382D/D470S a été mis en évidence. Son activité vis-à-vis d un aldéhyde modèle de configuration (2S) a été augmentée d un facteur 5 par rapport à l enzyme sauvage et la perte de l énantiosélectivité vis-à-vis desaldéhydes (2R) a été confirmée.Transketolase (TK, EC 2.2.1.1) catalyzes the formation of D-threo ketoses from (2R)-a-hydroxyaldehydes by the stereospecific formation of a C-C bond. Our aim was to invert the enantioselectivity of TK by protein engineering in order to obtain L-erytho ketoses (sought after for their potential pharmaceutical and/or nutritional applications) from (2S)-a-hydroxyaldehydes. For that purpose, a thermostable TK from thermophilic bacterium Geobacillus stearothermophilus (mTKgst) has been identified and overexpressed. After the study of the 3D-structure of mTKgst, two residues located in its active site (Leu382 and Asp470) were selected as mutation targets for the inversion of the enzyme s enantioselectivity. Both rational and semi-rational approaches were considered for the construction of the mutant mTKgst libraries. In the former, the two residues were modified by site-saturation mutagenesis. In the latter, short sequences of five amino acids, neighboring target ones, were modified using a cassette mutagenesis technique. A novel continuous pH-based assay has been developed for the high-throughput screening of the mTKgst libraries, using phenol red as pH indicator. The screening revealed mTKgst-L382D/D470S as the top mutant, showing a 5-fold activity improvement towards a model (2S)-hydroxyaldehyde and the loss of enantioselectivity towards the (2R)-aldehyde.CLERMONT FD-Bib.électronique (631139902) / SudocSudocFranceF

A pH-Based High-Throughput Assay for Transketolase: Fingerprinting of Substrate Tolerance and Quantitative Kinetics

International audienceA pH-based high-throughput assay method has been developed for the rapid and reliable measurement of transketolase (TK) activity. The method is based on the decarboxylation of lithium hydroxypyruvate (HPA) as a hydroxyacetyl donor with an aldehyde acceptor, using phenol red as the pH indicator. Upon release of carbon dioxide from HPA, the pH increase in the reaction mixture can be determined photometrically by the color change of the pH indicator. At low buffer concentration (2 mM triethanolamine, pH 7.5), the method is highly sensitive and allows continuous monitoring, for quantitative determination of the kinetic parameters. By using this method, the substrate specificities of the TK enzymes from Escherichia coli and Saccharomyces cerevisiae, as well as two active-site-modified variants of the E. coli TK (D469E, H26Y) were evaluated against a panel of substrate analogues; specific activities and kinetic constants could be rapidly determined. Substrate quality indicated by assay determination was substantiated with novel TK applications by using achiral 3-hydroxypropanal and 4-hydroxybutanal for preparative synthesis of chiral deoxyketose-type products. Determination of ee for the latter could be performed by chiral GC analysis, with an unambiguous correlation of the absolute configuration from rotation data. This pH-based assay method is broadly applicable and allows rapid, sensitive, and reliable screening of the substrate tolerance of known TK enzymes and variants obtained from directed evolution

A pH-Based High-Throughput Assay for Transketolase: Fingerprinting of Substrate Tolerance and Quantitative Kinetics

International audienceA pH-based high-throughput assay method has been developed for the rapid and reliable measurement of transketolase (TK) activity. The method is based on the decarboxylation of lithium hydroxypyruvate (HPA) as a hydroxyacetyl donor with an aldehyde acceptor, using phenol red as the pH indicator. Upon release of carbon dioxide from HPA, the pH increase in the reaction mixture can be determined photometrically by the color change of the pH indicator. At low buffer concentration (2 mM triethanolamine, pH 7.5), the method is highly sensitive and allows continuous monitoring, for quantitative determination of the kinetic parameters. By using this method, the substrate specificities of the TK enzymes from Escherichia coli and Saccharomyces cerevisiae, as well as two active-site-modified variants of the E. coli TK (D469E, H26Y) were evaluated against a panel of substrate analogues; specific activities and kinetic constants could be rapidly determined. Substrate quality indicated by assay determination was substantiated with novel TK applications by using achiral 3-hydroxypropanal and 4-hydroxybutanal for preparative synthesis of chiral deoxyketose-type products. Determination of ee for the latter could be performed by chiral GC analysis, with an unambiguous correlation of the absolute configuration from rotation data. This pH-based assay method is broadly applicable and allows rapid, sensitive, and reliable screening of the substrate tolerance of known TK enzymes and variants obtained from directed evolution

Engineering a Thermostable Transketolase for Unnatural Conversion of (2S)-Hydroxyaldehyde.

Special Issue sur invitation (Very Important Paper).International audienceTransketolase (TK) from various origins (including Escherichia coli and yeast) has been described to be fully enantiomer specific for (2R)-hydroxyaldehyde substrates. A thermostable TK from Geobacillus stearothermophilus (TKgst) was found to display a minor reactivity for (2S)-hydroxylated aldehydes. To improve this activity by directed protein evolution, we have built a library of TKgst variants by site saturation mutagenesis on two key positions L382 and D470. The best TKgst double mutant L382D/D470S shows up to 4- and 5-fold higher activities towards L-lactaldehyde and L-glyceraldehyde as acceptor substrates, respectively. Preparative utility of this mutant was demonstrated by the one-step synthesis of valuable L-ribulose and its 5-deoxy analogue with the L-erythro (3S,4S) configuration, which were previously inaccessible by using common TK sources

Thermostable Transketolase from Geobacillus stearothermophilus: Characterization and Catalytic Properties

International audienceHere we have characterized the first transketolase (TK) from a thermophilic microorganism, Geobacillus stearothermophilus, which was expressed from a synthetic gene in Escherichia coli. The G. stearothermophilus TK (mTKgst) retained 100% activity for one week at 50 8C and for 3 days at 658C, and has an optimum temperature range around 60- 70 8C, which will be useful for preparative applications and for future biocatalyst development. The thermostability of the mTKgst allowed us to carry out an easy, one-step purification by heat shock treatment of crude cell extracts at 65 8C for 45 min, directly yielding 132 mg of pure mTKgst from 1 L of culture. The reaction rate of mTKgst with glycolaldehyde was 14 times higher at 70 8C than at 20 8C, and 4 times higher at 50 8C when compared to E. coli TK under identical conditions. When tested at 50 8C with other aldehydes as acceptors, mTKgst activity was approximately 3 times higher than those obtained at 20 8C. Applications of this new TK in biocatalysis were performed with hydroxypyruvate as donor and three different aldehydes as acceptors - glycolaldehyde, d-glyceraldehyde and butyraldehyde - from which the corresponding products l-erythrulose 1, dxylulose 2 and 1,3-dihydroxyhexan-2-one 3 were obtained, respectively. The optical rotations for products 1 and 2 indicate that the stereospecificity of mTKgst is identical to that of other TK sources, leading to a (3S) configuration. With the non-hydroxylated substrate, butanal, the ee value was 85% (3S), showing higher enantioselectivity than the E. coli TK (75% ee, 3S). Processes at elevated temperatures could offer opportunities to extend the applications of TK biocatalysis, by favoring hydrophobic aldehyde acceptor substrate solubility and tolerance towards non-conventional media

Thermostable Transketolase from Geobacillus stearothermophilus: Characterization and Catalytic Properties

International audienceHere we have characterized the first transketolase (TK) from a thermophilic microorganism, Geobacillus stearothermophilus, which was expressed from a synthetic gene in Escherichia coli. The G. stearothermophilus TK (mTKgst) retained 100% activity for one week at 50 8C and for 3 days at 658C, and has an optimum temperature range around 60- 70 8C, which will be useful for preparative applications and for future biocatalyst development. The thermostability of the mTKgst allowed us to carry out an easy, one-step purification by heat shock treatment of crude cell extracts at 65 8C for 45 min, directly yielding 132 mg of pure mTKgst from 1 L of culture. The reaction rate of mTKgst with glycolaldehyde was 14 times higher at 70 8C than at 20 8C, and 4 times higher at 50 8C when compared to E. coli TK under identical conditions. When tested at 50 8C with other aldehydes as acceptors, mTKgst activity was approximately 3 times higher than those obtained at 20 8C. Applications of this new TK in biocatalysis were performed with hydroxypyruvate as donor and three different aldehydes as acceptors - glycolaldehyde, d-glyceraldehyde and butyraldehyde - from which the corresponding products l-erythrulose 1, dxylulose 2 and 1,3-dihydroxyhexan-2-one 3 were obtained, respectively. The optical rotations for products 1 and 2 indicate that the stereospecificity of mTKgst is identical to that of other TK sources, leading to a (3S) configuration. With the non-hydroxylated substrate, butanal, the ee value was 85% (3S), showing higher enantioselectivity than the E. coli TK (75% ee, 3S). Processes at elevated temperatures could offer opportunities to extend the applications of TK biocatalysis, by favoring hydrophobic aldehyde acceptor substrate solubility and tolerance towards non-conventional media