Thiamine‐Based Enzymes for Biotransformations

Pohl, Martina; Gocke, Dörte; Müller, Michael

doi:10.1002/9783527628698.hgc028

Cited by 9 publications

(14 citation statements)

References 168 publications

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“…The same is true for the following pairs: acetone and TCM, DMSO and i Prop, THF and dioxane, and EtOAc and dioxane. It can be concluded that the less polar solvent can better accumulate in the non-polar S -pocket, since it is mainly built of hydrophobic amino acid side chains4.…”

Section: Discussionmentioning

confidence: 99%

“…A well-known example is the carboligation of acetaldehyde and benzaldehyde catalyzed by pyruvate decarboxylases in fermenting yeasts yielding ( R )-1-hydroxy-1-phenylpropan-2-one (PAC), a precursor of ephedrine, with an ee >98% 3. In recent years, we have created a toolbox of ThDP-dependent enzymes including wild-type and engineered enzymes with varying selectivities to access a broad range of predominantly mixed aromatic and aliphatic chiral 2-hydroxy ketones 2f,4. In doing so, different organic cosolvents [e.g., dimethyl sulfoxide (DMSO),5 methyl tert -butyl ether (MTBE),6 2-methyltetrahydrofuran (MTHF)7] were used in order to increase the solubility of the aromatic compounds in the aqueous reaction system.…”

Section: Introductionmentioning

confidence: 99%

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Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

et al. 2012

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The asymmetric mixed carboligation of aldehydes with thiamine diphosphate (ThDP)-dependent enzymes is an excellent example where activity as well as changes in chemo- and stereoselectivity can be followed sensitively. To elucidate the influence of organic additives in enzymatic carboligation reactions of mixed 2-hydroxy ketones, we present a comparative study of six ThDP-dependent enzymes in 13 water-miscible organic solvents under equivalent reaction conditions. The influence of the additives on the stereoselectivity is most pronounced and follows a general trend. If the enzyme stereoselectivity in aqueous buffer is already >99.9% ee, none of the solvents reduces this high selectivity. In contrast, both stereoselectivity and chemoselectivity are strongly influenced if the enzyme is rather unselective in aqueous buffer. For the S-selective enzyme with the largest active site, we were able to prove a general correlation of the solvent-excluded volume of the additives with the effect on selectivity changes: the smaller the organic solvent molecule, the higher the impact of this additive. Further, a correlation to log P of the additives on selectivity was detected if two additives have almost the same solvent-excluded volume. The observed results are discussed in terms of structural, biochemical and energetic effects. This work demonstrates the potential of medium engineering as a powerful additional tool for varying enzyme selectivity and thus engineering the product range of biotransformations. It further demonstrates that the use of cosolvents should be carefully planned, as the solvents may compete with the substrate(s) for binding sites in the enzyme active site.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

et al. 2012

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“…An overview of all annotated sequences can be found in the ThDP‐dependent Enzyme Engineering Database (TEED, http://www.teed.uni-stuttgart.de) . Among these, enzymes catalysing the cleavage and formation of C–C bonds have been most intensively studied . Here, the potential to catalyse the formation of mixed carboligation products from a donor substrate and an acceptor substrate in a highly chemoselective and stereoselective manner is of special interest.…”

Section: Introductionmentioning

confidence: 99%

Engineering stereoselectivity of ThDP-dependent enzymes

Hailes

Rother

Müller

et al. 2013

FEBS J

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Thiamine diphosphate-dependent enzymes are broadly distributed in all organisms, and they catalyse a broad range of C-C bond forming and breaking reactions. Enzymes belonging to the structural families of decarboxylases and transketolases have been particularly well investigated concerning their substrate range, mechanism of stereoselective carboligation and carbolyase reaction. Both structurally different enzyme families differ also in stereoselectivity: enzymes from the decarboxylase family are predominantly R-selective, whereas those from the transketolase family are S-selective. In recent years a key focus of our studies has been on stereoselective benzoin condensation-like 1,2-additions. Meanwhile, several S-selective variants of pyruvate decarboxylase, benzoylformate decarboxylase and 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPH-CHC) synthase as well as R-selective transketolase variants were created that allow access to a broad range of enantiocomplementary a-hydroxyketones and a,a′-dihydroxyketones. This review covers recent studies and the mechanistic understanding of stereoselective C-C bond forming thiamine diphosphate-dependent enzymes, which has been guided by structure-function analyses based on mutagenesis studies and from influences of different substrates and organic co-solvents on stereoselectivity.

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“…While the decarboxylation of 2-ketoacids [10] and the carboligation of two aldehydes to 2-hydroxy ketones are catalysed by most members of the ThDP-dependent decarboxylases [9], their substrate ranges are different. The well characterised PDC from Saccharomyces cerevisiae , BFD from Pseudomonas putida and BAL from Pseudomonas fluorescence accept a broad variety of substrates [7,11,12], while SEPHCHC-synthase (MenD) is limited to a small number of substrates [13,14]. Additional complexity of C-C bond formation results from the fact that a substrate might be either a donor, which is activated by addition to ThDP in the active site, or an acceptor, which reacts with the ThDP-bound donor, resulting in different products [7,11,12].…”

Section: Introductionmentioning

confidence: 99%

“…The well characterised PDC from Saccharomyces cerevisiae , BFD from Pseudomonas putida and BAL from Pseudomonas fluorescence accept a broad variety of substrates [7,11,12], while SEPHCHC-synthase (MenD) is limited to a small number of substrates [13,14]. Additional complexity of C-C bond formation results from the fact that a substrate might be either a donor, which is activated by addition to ThDP in the active site, or an acceptor, which reacts with the ThDP-bound donor, resulting in different products [7,11,12]. Reactions catalysed by members of the structural group of ThDP-dependent decarboxylases include decarboxylation of 2-keto acids, synthesis of various chiral 2-hydroxy ketones by asymmetric benzoin- [11,15] and cross-benzoin condensation [16,17], the racemic resolution of 2-hydroxy ketones via C-C bond cleavage [18], and Stetter-like reactions, e.g.…”

Section: Introductionmentioning

confidence: 99%

A standard numbering scheme for thiamine diphosphate-dependent decarboxylases

et al. 2012

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BackgroundStandard numbering schemes for families of homologous proteins allow for the unambiguous identification of functionally and structurally relevant residues, to communicate results on mutations, and to systematically analyse sequence-function relationships in protein families. Standard numbering schemes have been successfully implemented for several protein families, including lactamases and antibodies, whereas a numbering scheme for the structural family of thiamine-diphosphate (ThDP) -dependent decarboxylases, a large subfamily of the class of ThDP-dependent enzymes encompassing pyruvate-, benzoylformate-, 2-oxo acid-, indolpyruvate- and phenylpyruvate decarboxylases, benzaldehyde lyase, acetohydroxyacid synthases and 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD) is still missing.Despite a high structural similarity between the members of the ThDP-dependent decarboxylases, their sequences are diverse and make a pairwise sequence comparison of protein family members difficult.ResultsWe developed and validated a standard numbering scheme for the family of ThDP-dependent decarboxylases. A profile hidden Markov model (HMM) was created using a set of representative sequences from the family of ThDP-dependent decarboxylases. The pyruvate decarboxylase from S. cerevisiae (PDB: 2VK8) was chosen as a reference because it is a well characterized enzyme. The crystal structure with the PDB identifier 2VK8 encompasses the structure of the ScPDC mutant E477Q, the cofactors ThDP and Mg2+ as well as the substrate analogue (2S)-2-hydroxypropanoic acid. The absolute numbering of this reference sequence was transferred to all members of the ThDP-dependent decarboxylase protein family. Subsequently, the numbering scheme was integrated into the already established Thiamine-diphosphate dependent Enzyme Engineering Database (TEED) and was used to systematically analyze functionally and structurally relevant positions in the superfamily of ThDP-dependent decarboxylases.ConclusionsThe numbering scheme serves as a tool for the reliable sequence alignment of ThDP-dependent decarboxylases and the unambiguous identification and communication of corresponding positions. Thus, it is the basis for the systematic and automated analysis of sequence-encoded properties such as structural and functional relevance of amino acid positions, because the analysis of conserved positions, the identification of correlated mutations and the determination of subfamily specific amino acid distributions depend on reliable multisequence alignments and the unambiguous identification of the alignment columns. The method is reliable and robust and can easily be adapted to further protein families.

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Thiamine‐Based Enzymes for Biotransformations

Cited by 9 publications

References 168 publications

Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

Engineering stereoselectivity of ThDP-dependent enzymes

A standard numbering scheme for thiamine diphosphate-dependent decarboxylases

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