Publications by authors named "Laurence Hecquet"

Vibrio vulnificus (vv) is a multidrug-resistant human bacterial pathogen whose prevalence is expected to increase over the years. Transketolases (TK), transferases catalyzing two reactions of the nonoxidative branch of the pentose-phosphate pathway and therefore linked to several crucial metabolic pathways, are potential targets for new drugs against this pathogen. Here, the vvTK is crystallized and its structure is solved at 2.

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Article Synopsis
  • Thiamine diphosphate-dependent enzymes, especially transketolases, are essential for creating specific carbon-carbon bonds that produce valuable hydroxyketones.
  • A straightforward colorimetric assay based on pH changes was developed for quickly assessing the activity of transketolases, streamlining their screening process.
  • The assay utilizes bromothymol blue as a pH indicator to visually indicate active transketolase variants without the need for enzyme extraction.
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A computational model for human transketolase was proposed, showing that thiamine diphosphate activation was based on His110 in place of His481 reported in yeast transketolase. In addition, a complete catalytic reaction pathway was investigated using d-xylulose-5-phosphate and d-ribose-5-phosphate as substrates, showing at every step a perfect superimposition of our model with high-resolution crystallographic structures 3MOS, 4KXV, and 4KXX. This study shows that H2N4' of the active thiamine diphosphate "V form" no longer has a self-activating role but allows self-stabilization of the cofactor and of the Breslow intermediate.

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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.

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Enzymes catalyzing asymmetric carboligation reactions typically show very high substrate specificity for their nucleophilic donor substrate components. Structure-guided engineering of the thermostable transketolase from Geobacillus stearothermophilus by directed in vitro evolution yielded new enzyme variants that are able to utilize pyruvate and higher aliphatic homologues as nucleophilic components for acyl transfer instead of the natural polyhydroxylated ketose phosphates or hydroxypyruvate. The single mutant H102T proved the best hit toward 3-methyl-2-oxobutyrate as donor, while the double variant H102L/H474S showed highest catalytic efficiency toward pyruvate as donor.

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In the present work, we describe a new thiamine amperometric biosensor based on thiamine pyrophosphate (ThDP)-dependent transketolase (TK)-catalyzed reaction, followed by the oxidative trapping of TK intermediate α,β-dihydroxyethylthiamine diphosphate (DHEThDP) within the enzymatic active site. For the biosensor design purpose, TK from Escherichia coli (TKec) was immobilized in MgAl-NO Layered Double Hydroxides (LDH) and the electrochemical detection was achieved with the TKec/LDH modified glassy carbon electrode (GCE). The transduction process was based on the ability of Fe(CN) to oxidize DHEThDP to glycolic acid along with ThDP regeneration.

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We propose the first computational model for transketolase (TK), a thiamine diphosphate (ThDP)-dependent enzyme, using a quantum mechanical/molecular mechanical method on the basis of crystallographic TK structures from yeast and Escherichia coli, together with experimental kinetic data reported in the literature with wild-type and mutant TK. This model allowed us to define a new route for ThDP activation in the enzyme environment. We evidenced a strong interaction between ThDP and Glu418B of the TK active site, itself stabilized by Glu162A.

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Article Synopsis
  • - Researchers used directed evolution to enhance the thermostable transketolase enzyme from Geobacillus stearothermophilus.
  • - They implemented a colorimetric screening method based on pH to identify effective mutants.
  • - The evolved mutants showed up to 16 times greater activity with aliphatic aldehydes and demonstrated high enantioselectivity (over 95% ee) in a key reaction step.
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This paper describes an innovative amperometric biosensor for the in vitro determination of activity of transketolase from Escherichia coli (TKec) using commercially available TK substrates, namely d-fructose-6-phosphate a physiological donor and glycolaldehyde the best non-phosphorylated acceptor. A galactose oxidase (GAOx) biosensor, based on the immobilization of this enzyme within laponite clay, allows amperometric detection of L-erythrulose released upon TK-catalyzed reaction. A calibration curve has been established from 0.

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Article Synopsis
  • The study focused on immobilizing TKec, an enzyme from Escherichia coli, on a specific structure called MgAl-NO3 layered double hydroxides (LDH) using two methods: adsorption and coprecipitation.
  • Different pH conditions were tested for adsorption, while coprecipitation was done with the enzyme present during LDH formation.
  • The resulting biohybrids were analyzed for stability and enzymatic activity, revealing that the coprecipitated version (TKec@MgAl-Gly-Gly) had the highest activity (12 U/mg) and thin films of this biohybrid showed consistent activity across multiple tests.
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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.

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This paper proposes a new concept of transketolase (TK) activity profiling. A tyrosinase (PPO) biosensor, based on the immobilization of this enzyme in a Mg(2)Al-Cl layered double hydroxide, was developed for the amperometric detection of N-acetyl-l-tyrosine ethyl ester monohydrate (N-Ac-Tyr-OEt) at -0.2V.

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Dihydroxyacetone phosphate (DHAP) was synthesized in high purity and yield in four steps starting from dihydroxyacetone dimer (DHA) (47% overall yield). DHA was converted into 2,2-dimethoxypropane-1,3-diol, which was desymmetrized by acetylation with lipase AK. The alcohol function was phosphorylated to give dibenzyl phosphate ester 4.

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2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase is a key enzyme in the Entner-Doudoroff pathway of bacteria. It catalyzes the reversible production of KDPG from pyruvate and D-glyceraldehyde 3-phosphate through a class I Schiff base mechanism. On the basis of aldolase mechanistic pathway, various pyruvate analogues bearing beta-diketo structures were designed and synthesized as potential inhibitors.

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