Characterisation of a hyperthermophilic transketolase from Thermotoga maritima DSM3109 as a biocatalyst for 7-keto-octuronic acid synthesis

Max Cárdenas-Fernández; Fabiana Subrizi; Dragena Dobrijevic; Helen C. Hailes; John M.  Ward

doi:10.1039/d1ob01237a

Characterisation of a hyperthermophilic transketolase from Thermotoga maritima DSM3109 as a biocatalyst for 7-keto-octuronic acid synthesis

Max Cárdenas-Fernández
, Fabiana Subrizi
, Dragena Dobrijevic
, Helen C. Hailes
, John M. Ward

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

14 Citations (SciVal)

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Abstract

Transketolase (TK) is a fundamentally important enzyme in industrial biocatalysis which carries out a stereospecific carbon–carbon bond formation, and is widely used in the synthesis of prochiral ketones. This study describes the biochemical and molecular characterisation of a novel and unusual hyperthermophilic TK from Thermotoga maritima DSM3109 (TKtmar). TKtmar has a low protein sequence homology compared to the already described TKs, with key amino acid residues in the active site highly conserved. TKtmar has a very high optimum temperature (>90 °C) and shows pronounced stability at high temperature (e.g. t1/2 99 and 9.3 h at 50 and 80 °C, respectively) and in presence of organic solvents commonly used in industry (DMSO, acetonitrile and methanol). Substrate screening showed activity towards several monosaccharides and aliphatic aldehydes. In addition, for the first time, TK specificity towards uronic acids was achieved with TKtmar catalysing the efficient conversion of D-galacturonic acid and lithium hydroxypyruvate into 7-keto-octuronic acid, a very rare C8 uronic acid, in high yields (98%, 49 mM).

Original language	English
Pages (from-to)	6493-6500
Number of pages	8
Journal	Organic and Biomolecular Chemistry
Issue number	29
Early online date	7 Jul 2021
DOIs	https://doi.org/10.1039/d1ob01237a
Publication status	Published - 7 Aug 2021

Bibliographical note

Funding

M. Cárdenas-Fernández and J. M. Ward would like to thank the UK Biotechnology and Biological Sciences Research Council (BBSRC) for financial support of this work with BB/R021627/1 (ERA CoBioTech Grant). F. Subrizi and H. C. Hailes would like to thank the financial support from BBSRC BB/R021643/1 (ERACoBioTech Grant). Input and advice from the project collaborators from the ERABioCat European Project TRALAMINOL is also acknowledged.

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10.1039/d1ob01237aLicence: CC BY 3.0

Characterisation of a hyperthermophilic transketolase from Thermotoga maritima DSM3109 as a biocatalyst for 7-keto-octuronic acid synthesis
Copyright © The Royal Society of Chemistry 2021. Open Access Article. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.
Final published version, 988 KBLicence: CC BY 3.0

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title = "Characterisation of a hyperthermophilic transketolase from Thermotoga maritima DSM3109 as a biocatalyst for 7-keto-octuronic acid synthesis",

abstract = "Transketolase (TK) is a fundamentally important enzyme in industrial biocatalysis which carries out a stereospecific carbon–carbon bond formation, and is widely used in the synthesis of prochiral ketones. This study describes the biochemical and molecular characterisation of a novel and unusual hyperthermophilic TK from Thermotoga maritima DSM3109 (TKtmar). TKtmar has a low protein sequence homology compared to the already described TKs, with key amino acid residues in the active site highly conserved. TKtmar has a very high optimum temperature (>90 °C) and shows pronounced stability at high temperature (e.g. t1/2 99 and 9.3 h at 50 and 80 °C, respectively) and in presence of organic solvents commonly used in industry (DMSO, acetonitrile and methanol). Substrate screening showed activity towards several monosaccharides and aliphatic aldehydes. In addition, for the first time, TK specificity towards uronic acids was achieved with TKtmar catalysing the efficient conversion of D-galacturonic acid and lithium hydroxypyruvate into 7-keto-octuronic acid, a very rare C8 uronic acid, in high yields (98\%, 49 mM).",

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AB - Transketolase (TK) is a fundamentally important enzyme in industrial biocatalysis which carries out a stereospecific carbon–carbon bond formation, and is widely used in the synthesis of prochiral ketones. This study describes the biochemical and molecular characterisation of a novel and unusual hyperthermophilic TK from Thermotoga maritima DSM3109 (TKtmar). TKtmar has a low protein sequence homology compared to the already described TKs, with key amino acid residues in the active site highly conserved. TKtmar has a very high optimum temperature (>90 °C) and shows pronounced stability at high temperature (e.g. t1/2 99 and 9.3 h at 50 and 80 °C, respectively) and in presence of organic solvents commonly used in industry (DMSO, acetonitrile and methanol). Substrate screening showed activity towards several monosaccharides and aliphatic aldehydes. In addition, for the first time, TK specificity towards uronic acids was achieved with TKtmar catalysing the efficient conversion of D-galacturonic acid and lithium hydroxypyruvate into 7-keto-octuronic acid, a very rare C8 uronic acid, in high yields (98%, 49 mM).

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Characterisation of a hyperthermophilic transketolase from Thermotoga maritima DSM3109 as a biocatalyst for 7-keto-octuronic acid synthesis

Abstract

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