Fluorogenic substrates for the screening assay of transketolase through beta-elimination of umbelliferone—Development, scope and limitations

Charmantray, Franck; Légeret, Bertrand; Hélaine, Virgil; Hecquet, Laurence

doi:10.1016/j.jbiotec.2009.12.022

Cited by 6 publications

(3 citation statements)

References 33 publications

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“…[23] An umbelliferone-based sensitive fluorogenic assay has been devised to determine TK activity and stereoselectivity. [24] Although operated in a continuous mode, like the standard method, none of these highly substrate-specific methods is adaptable to probe the kinetics of TK for nonnatural substrate analogues.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2012

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A 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.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2012

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“…Initially, TK activity can be measured either by spectrophotometry by using NADH-dependent auxiliary enzymes 23,24 , chromophores, such as phenol red used in a pH-based high throughput assay 25 or as tetrazolium red which oxidizes non-α-hydroxylated substrates 26 , or by fluorescence using modified substrates. 27 However, these optical methods require a secondary enzyme and are limited by a lack of sensitivity and specificity or by the harsh synthesis of unnatural fluorescent substrates. Another spectrophotometric method based on the reduction of potassium ferricyanide by the DHETPP intermediate was also described.…”

mentioning

confidence: 99%

“…In order to find some new efficient TK inhibitors, a rapid and sensitive enzymatic assay is a prerequisite. Initially, TK activity can be measured either by spectrophotometry by using NADH-dependent auxiliary enzymes , , chromophores, such as phenol red used in a pH-based high throughput assay or as tetrazolium red which oxidizes non-α-hydroxylated substrates, or by fluorescence using modified substrates . However, these optical methods require a secondary enzyme and are limited by a lack of sensitivity and specificity or by the harsh synthesis of unnatural fluorescent substrates.…”

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confidence: 99%

Innovative Electrochemical Screening Allows Transketolase Inhibitors to Be Identified

et al. 2018

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Transketolases (TKs) are ubiquitous thiamine pyrophosphate (TPP)-dependent enzymes of the nonoxidative branch of the pentose phosphate pathway. They are considered as interesting therapeutic targets in numerous diseases and infections (e.g., cancer, tuberculosis, malaria), for which it is important to find specific and efficient inhibitors. Current TK assays require important amounts of enzyme, are time-consuming, and are not specific. Here, we report a new high throughput electrochemical assay based on the oxidative trapping of the TK-TPP intermediate. After electrode characterization, the enzyme loading, electrochemical protocol, and substrate concentration were optimized. Finally, 96 electrochemical assays could be performed in parallel in only 7 min, which allows a rapid screening of TK inhibitors. Then, 1360 molecules of an in-house chemical library were screened and one early lead compound was identified to inhibit TK from E. coli with an IC of 63 μM and an inhibition constant ( K) of 3.4 μM. The electrochemical assay was also used to propose an inhibition mechanism.

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New Applications of Transketolase: Cascade Reactions for Assay Development

Hecquet

Fessner

Hélaine

et al. 2014

Cascade Biocatalysis

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Fluorogenic substrates for the screening assay of transketolase through beta-elimination of umbelliferone—Development, scope and limitations

Cited by 6 publications

References 33 publications

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

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

Innovative Electrochemical Screening Allows Transketolase Inhibitors to Be Identified

New Applications of Transketolase: Cascade Reactions for Assay Development

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