Engineering a thermostable transketolase for unnatural conversion of (2S)-hydroxyaldehydes

Juliane Abdoul Zabar; Marion Lorillière; Dong Yi; Saravanan Thangavelu; Titu Devamani; Lionel Nauton; Franck Charmantray; Virgil Hélaine; Wolf Dieter Fessner; Laurence Hecquet

doi:10.1002/adsc.201500207

Engineering a thermostable transketolase for unnatural conversion of (2S)-hydroxyaldehydes

Juliane Abdoul Zabar
, Marion Lorillière
, Dong Yi
, Saravanan Thangavelu
, Titu Devamani
, Lionel Nauton
, Franck Charmantray
, Virgil Hélaine
, Wolf Dieter Fessner
, Laurence Hecquet

Biotechnology Institute

Research output: Contribution to journal › Article › peer-review

30 Scopus citations

Abstract

Transketolase (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 (TK_gst) 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 TK_gst variants by site saturation mutagenesis on two key positions L382 and D470. The best TK_gst 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.

Original language	English (US)
Pages (from-to)	1715-1720
Number of pages	6
Journal	Advanced Synthesis and Catalysis
Volume	357
Issue number	8
DOIs	https://doi.org/10.1002/adsc.201500207
State	Published - May 1 2015

Bibliographical note

Publisher Copyright:
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Keywords

carboligation
mutagenesis
stereoselectivity
transketolase
α-hydroxylated aldehydes

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10.1002/adsc.201500207

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title = "Engineering a thermostable transketolase for unnatural conversion of (2S)-hydroxyaldehydes",

abstract = "Transketolase (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.",

keywords = "carboligation, mutagenesis, stereoselectivity, transketolase, α-hydroxylated aldehydes",

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year = "2015",

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doi = "10.1002/adsc.201500207",

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T1 - Engineering a thermostable transketolase for unnatural conversion of (2S)-hydroxyaldehydes

AU - Abdoul Zabar, Juliane

AU - Lorillière, Marion

AU - Yi, Dong

AU - Thangavelu, Saravanan

AU - Devamani, Titu

AU - Nauton, Lionel

AU - Charmantray, Franck

AU - Hélaine, Virgil

AU - Fessner, Wolf Dieter

AU - Hecquet, Laurence

PY - 2015/5/1

Y1 - 2015/5/1

N2 - Transketolase (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.

AB - Transketolase (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.

KW - carboligation

KW - mutagenesis

KW - stereoselectivity

KW - transketolase

KW - α-hydroxylated aldehydes

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JO - Advanced Synthesis and Catalysis

JF - Advanced Synthesis and Catalysis

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ER -

Engineering a thermostable transketolase for unnatural conversion of (2S)-hydroxyaldehydes

Abstract

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