A thermostable transketolase evolved for aliphatic aldehyde acceptors

Dong, Yi; Saravanan, T.; Devamani, Titu; Charmantray, Franck; Hecquet, Laurence; Fessner, Wolf Dieter

doi:10.1039/c4cc08436e

Cited by 37 publications

(68 citation statements)

References 32 publications

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“…Among further residues, which were identified to influence stereoselectivity, is phenylalanine in standard position 434 found in Ec TK, Gst TK and Sc TK and conserved with 62 % in the whole TK superfamily . Also leucine in standard position 382 is conserved among these TKs and found in 61 % of all sequences of the TK superfamily. Besides, an isoleucine found in Ec TK and Sc TK in standard position 189 is replaced by L191 in Gst TK .…”

Section: Resultsmentioning

confidence: 99%

“…Also leucine in standard position 382 is conserved among these TKs and found in 61 % of all sequences of the TK superfamily. Besides, an isoleucine found in Ec TK and Sc TK in standard position 189 is replaced by L191 in Gst TK . Aspartate in standard position 469 plays a role in substrate binding, activity enhancement and stereoselectivity, is conserved among the three TKs mentioned above and found in 59 % of TK sequences.…”

Section: Resultsmentioning

confidence: 99%

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Towards a Mechanistic Understanding of Factors Controlling the Stereoselectivity of Transketolase

et al. 2018

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A structural model for thiamine‐diphosphate (ThDP)‐dependent transketolase (TK) was developed to analyse the effect of amino acid exchanges on the stereoselectivity of this synthetically important class of enzymes. In this study the carboligation of 3‐hydroxypyruvate as a donor and propanal, as well as pentanal, was studied. Based on literature data and additional mutagenesis studies using E. coli TK, a four‐state model was developed to explain the stereoselectivity of TKs by the relative orientation of donor and acceptor substrates in the active site prior to C−C‐bond formation. To enable a functional comparison of relevant amino acids of TKs from different species, a standard numbering scheme was developed. Using this concept, H26, H261, and F434 were identified as the key residues which mediate stereoselectivity, where two main factors influenced the arrangement of ThDP‐bound donor and acceptor prior to carboligation: the relative orientation of the substrate side chains and the orientation of the acceptor carbonyl group towards the donor hydroxy group. This model provides a first framework to understand the structure‐function relationships of TKs with respect to their stereoselectivity.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Towards a Mechanistic Understanding of Factors Controlling the Stereoselectivity of Transketolase

et al. 2018

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“…A comprehensive comparative analysis of the substrate tolerance of TK Gst showed that non‐α‐hydroxylated aldehydes are accepted by wild‐type TK Gst with activity 40 times lower than that towards the corresponding α‐hydroxylated derivatives (Scheme ) . The removal of the α‐hydroxy group reduces the number of possible hydrogen‐bonding contacts between TK and acceptor substrate and might also affect the catalytic efficiency and the stereoselectivity of TK Gst . Thus, a structural adjustment based on mutagenesis of the residues in the active site that make contact with the substrate might help to improve the binding and activation of non‐α‐hydroxylated substrates.…”

Section: Methodsmentioning

confidence: 99%

“…At the position opposite Asp470, Leu382 provides a hydrophobic contribution to substrate binding. Therefore, in our previous study, these two key residues were chosen as the preferred mutagenesis positions for the creation of a double‐site saturation mutagenesis library, out of which the top variant—L382F/D470—has specific activity towards propanal 11 times higher than that of the wild type, yielding the 3 S product with high ee (>95 %) . However, all of the positive variants from this library were found to maintain the stereopreference for the 3 S configuration, except for the variant L382I/D470L, which showed inversed stereoselectivity but only with a very low ee (9.4 %) .…”

Section: Methodsmentioning

confidence: 99%

Second‐Generation Engineering of a Thermostable Transketolase (TK_Gst) for Aliphatic Aldehyde Acceptors with Either Improved or Reversed Stereoselectivity

et al. 2017

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The transketolase from Geobacillus stearothermophilus (TK ) is a thermostable enzyme with notable high activity and stability at elevated temperatures, but it accepts non-α-hydroxylated aldehydes only with low efficiency. Here we report a protein engineering study of TK based on double-site saturation mutagenesis either at Leu191 or at Phe435 in combination with Asp470; these are the residues responsible for substrate binding in the active site. Screening of the mutagenesis libraries resulted in several positive variants with activity towards propanal up to 7.4 times higher than that of the wild type. Variants F435L/D470E and L191V/D470I exhibited improved (73 % ee, 3S) and inverted (74 % ee, 3R) stereoselectivity, respectively, for propanal. L191V, L382F/E, F435L, and D470/D470I were concluded to be positive mutations at Leu191, Leu382, Phe435, and Asp470 both for activity and for stereoselectivity improvement. These results should benefit further engineering of TK for various applications in asymmetric carboligation.

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“…For applications in biocatalysis, lithium hydroxypyruvate (HPA) is used as the donor substrate because the reaction becomes irreversible by the decarboxylation of HPA and the release of bicarbonate ions. Since TKs from different microbial sources can accept a wide range of hydroxylated and non-hydroxylated aliphatic, [4][5][6][7] and cyclic and aromatic aldehydes, 4,8 they represent a highly attractive biocatalytic platform technology for the synthesis of chiral polyols, (3S)-ketoses, with an ever-increasing range of applications [8][9][10][11][12] (Fig. 1).…”

Section: Introductionmentioning

confidence: 99%

Droplet millifluidics for kinetic study of transketolase

et al. 2016

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We present a continuous-flow reactor at the millifluidic scale coupled with an online, non-intrusive spectroscopic monitoring method for determining the kinetic parameters of an enzyme, transketolase (TK) used in biocatalysis for the synthesis of polyols by carboligation. The millifluidic system used is based on droplet flow, a well-established method for kinetic chemical data acquisition. The TK assay is based on the direct quantitative measurement of bicarbonate ions released during the transketolase-catalysed reaction in the presence of hydroxypyruvic acid as the donor, thanks to an irreversible reaction: bicarbonate ions react with phosphoenolpyruvate (PEP) in the presence of PEP carboxylase as the first auxiliary enzyme. The oxaloacetate formed is reduced to malate by NADH in the reaction catalysed by malate dehydrogenase as the second auxiliary enzyme. The extent of oxidation of NADH was measured by spectrophotometry at 340 nm. This system gives a direct, quantitative, generic method to evaluate the TK activity versus different substrates. We demonstrate the accuracy of this strategy to determine the enzymatic kinetic parameters and to study the substrate specificity of a thermostable TK from thermophilic microorganism Geobacillus stearothermophilus, offering promising prospects in biocatalysis. Millifluidic systems are useful in this regard as they can be used to rapidly evaluate the TK activity towards various substrates, and also different sets of conditions, identifying the optimal operating environment while minimizing resource consumption and ensuring high control over the operating conditions. Published by AIP Publishing. [http://dx

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A thermostable transketolase evolved for aliphatic aldehyde acceptors

Cited by 37 publications

References 32 publications

Towards a Mechanistic Understanding of Factors Controlling the Stereoselectivity of Transketolase

Towards a Mechanistic Understanding of Factors Controlling the Stereoselectivity of Transketolase

Second‐Generation Engineering of a Thermostable Transketolase (TK_Gst) for Aliphatic Aldehyde Acceptors with Either Improved or Reversed Stereoselectivity

Droplet millifluidics for kinetic study of transketolase

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A thermostable transketolase evolved for aliphatic aldehyde acceptors

Cited by 37 publications

References 32 publications

Towards a Mechanistic Understanding of Factors Controlling the Stereoselectivity of Transketolase

Towards a Mechanistic Understanding of Factors Controlling the Stereoselectivity of Transketolase

Second‐Generation Engineering of a Thermostable Transketolase (TKGst) for Aliphatic Aldehyde Acceptors with Either Improved or Reversed Stereoselectivity

Droplet millifluidics for kinetic study of transketolase

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Second‐Generation Engineering of a Thermostable Transketolase (TK_Gst) for Aliphatic Aldehyde Acceptors with Either Improved or Reversed Stereoselectivity