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Transketolases (TKs) are key enzymes of the pentose phosphate pathway, regulating several other critical pathways in cells. Considering their metabolic importance, TKs are expected to be conserved throughout evolution. However, Tittmann et al. ( J Biol Chem , 2010 , 285(41): 31559-31570) demonstrated that Homo sapiens TK ( hs TK) possesses several structural and kinetic differences compared to bacterial TKs. Here, we study 14 TKs from pathogenic bacteria, fungi, and parasites and compare them with hs TK using biochemical, bioinformatic, and structural approaches. For this purpose, six new TK structures are solved by X-ray crystallography, including the TK of Plasmodium falciparum . All of these TKs have the same general fold as bacterial TKs. This comparative study shows that hs TK greatly differs from TKs from pathogens in terms of enzymatic activity, spatial positions of the active site, and monomer-monomer interface residues. An ubiquitous structural pattern is identified in all TKs as a six-residue histidyl crown around the TK cofactor (thiamine pyrophosphate), except for hs TK containing only five residues in the crown. Residue mapping of the monomer-monomer interface and the active site reveals that hs TK contains more unique residues than other TKs. From an evolutionary standpoint, TKs from animals (including H. sapiens ) and Schistosoma sp. belong to a distinct structural group from TKs of bacteria, plants, fungi, and parasites, mostly based on a different linker between domains, raising hypotheses regarding evolution and regulation.

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 2.1 Å. A crown of 6 histidyl residues is observed in the active site and expected to participate in the thiamine pyrophosphate (cofactor) activation. Docking of fructose-6-phosphate and ferricyanide used in the activity assay, suggests that both substrates can bind vvTK simultaneously. This is confirmed by steady-state kinetics showing a sequential mechanism, on the contrary to the natural transferase reaction which follows a substituted mechanism. Inhibition by the I38-49 inhibitor (2-(4-ethoxyphenyl)-1-(pyrimidin-2-yl)-1H-pyrrolo[2,3-b]pyridine) reveals for the first time a cooperative behavior of a TK and docking experiments suggest a previously undescribed binding site at the interface between the pyrophosphate and pyridinium domains., (© 2023 The Protein Society.)

Thiamine diphosphate-dependent enzymes, and specifically transketolases, form one of the most important families of biocatalytic tools for enantioselective carbon-carbon bond formation yielding various hydroxyketones of biological interest. To enable substrate profiling of transketolases for acceptance of different donors and acceptors, a simple, direct colorimetric assay based on pH reaction variation was developed to establish a high-throughput solid-phase assay. This assay reduces the screening effort in the directed evolution of transketolases, as only active variants are selected for further analysis. Transketolase activity is detected as bicarbonate anions released from the α-ketoacid donor substrate, which causes the pH to rise. A pH indicator, bromothymol blue, which changes color from yellow to blue in alkaline conditions, was used to directly detect, with the naked eye, clones expressing active transketolase variants, obviating enzyme extraction., (© 2021 Wiley-VCH GmbH.)

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. These advances in our knowledge of the human transketolase mechanism offer interesting prospects for the design of new drugs, this enzyme being involved in several diseases, and for a better understanding of the reactions catalyzed by transketolases from other sources.

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 50 of 63 μM and an inhibition constant ( K I ) of 3.4 μM. The electrochemical assay was also used to propose an inhibition mechanism.

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. The latter variant was able to complement the auxotrophic deficiency of Escherichia coli cells arising from a deletion of the dxs gene, which encodes for activity of the first committed step into the terpenoid biosynthesis, offering the chance to employ a growth selection test for further enzyme optimization., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The transketolase from Geobacillus stearothermophilus (TK Gst ) 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 Gst 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 Gst for various applications in asymmetric carboligation., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 Mg 2 Al-NO 3 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) 6 3- was re-oxidized at +0.5V vs Ag-AgCl and the reaction was followed by chronoamperometry. The TKec/LDH/GCE biosensor was optimized using the best TK donor substrates, namely l-erythrulose and d-fructose-6-phosphate. ThDP was assayed with great sensitivity (3831mAM 6 4- was re-oxidized at +0.5V vs Ag-AgCl and the reaction was followed by chronoamperometry. The TKec/LDH/GCE biosensor was optimized using the best TK donor substrates, namely l-erythrulose and d-fructose-6-phosphate. ThDP was assayed with great sensitivity (3831mAM -1 cm -2 ) over 20-400nM linear range., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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. The crucial point highlighted here is that deprotonation of ThDP C2 is not performed by ThDP N4' as reported in the literature, but by His481B, involving a HOH688A molecule bridge. Thus, ThDP N4' is converted from an amino form to an iminium form, ensuring the stabilization of the C2 carbanion or carbene. Finally, ThDP activation proceeds via an intermolecular process and not by an intramolecular one as reported in the literature. More generally, this proposed ThDP activation mechanism can be applied to some other ThDP-dependent enzymes and used to define the entire TK mechanism with donor and acceptor substrates more accurately.

Directed evolution of the thermostable transketolase from Geobacillus stearothermophilus based on a pH-based colorimetric screening of smart libraries yielded several mutants with up to 16-fold higher activity for aliphatic aldehydes and high enantioselectivity (>95% ee) in the asymmetric carboligation step.

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 0.01 to 0.1 U ml(-1) TKec concentration in solution. These data are comparable to that obtained by a fluorometric method. In order to ensure a higher sensitivity and re-usability of the system, an original bienzymatic sensing system was further developed based on apoenzyme TKec and GAOx separately immobilized on the electrode surface. The inner sensing layer contains GAOx@laponite and the outer layer TKec@layered double hydroxide biohybrid. The biosensor response was validated by the determination of KD(app) for thiamine diphosphate, the TK cofactor and the inhibition action of two commercially available products, pyrophosphate, a TK cofactor analog and d-arabinose-5-phosphate, a substrate analog., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Immobilization of TK from Escherichia coli (TKec) on MgAl-NO3 layered double hydroxides (LDH) was carried out by two processes: adsorption and coprecipitation. As a comparison, the adsorption method was realized either at pH 7.5 in buffered solutions (MOPS and Gly-Gly) or in pure water. For the coprecipitation method, the formation of the inorganic LDH support was realized directly in the presence of TKec solubilized in Gly-Gly. The prepared biohybrids, called respectively TKec@LDHads and TKec@LDHcop, were characterized by powder X-ray diffraction, FTIR spectroscopy in comparison with TKec free reference products, i.e. MgAl-NO3, MgAl-Gly-Gly. The enzymatic activities of the various TKec@LDH biohybrids as well as their stabilities over time were investigated by UV-vis assay. A maximum of activity (12 U/mg of solid) was reached for TKec@MgAl-Gly-Gly biohybrid prepared by coprecipitation. Finally, thin films were prepared through a one-step deposition on a polished support. The enzymatic activity of the resulting TKec@MgAl-Gly-Glycop film was tested over four recycling processes with a reproducible activity of 2.7 U/mg cm(2)., (© 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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. This compound was released during an enzymatic reaction catalyzed by TK with N-acetyl-O-(2R, 3S, 5-trihydroxy-4-oxopentyl)-l-tyrosine ethyl ester used as donor substrate. This tyrosinase biosensor was optimized for the detection of TK activity, including PPO optimum substrate concentration, electrolyte nature, pH, and influence of bovine serum albumin (BSA). It was found that N-Ac-Tyr-OEt release is dependent on TK concentration (U/mL) in the electrolyte medium. These results demonstrate the sensitivity and specificity of the tyrosinase biosensor designed for in vitro detection of TK activity, which is known to be involved in several diseases., (Copyright 2010 Elsevier B.V. All rights reserved.)

Probes were developed for the in vivo detection of transketolase activity by the use of a complementation assay in Escherichia coli auxotrophs They combine the d-threo ketose moiety recognised by transketolase and the side chain of leucine or methionine. These compounds were donor substrates of yeast transketolase leading to the release of the corresponding alpha-hydroxyaldehydes which could be converted in E. coli by a cascade of reactions into leucine or methionine required for cellular growth.

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. From 4, two routes were investigated for large-scale synthesis of DHAP. First, acetate hydrolysis was performed prior to hydrogenolysis of the phosphate protective groups. The acetal hydrolysis was finally catalyzed by the phosphate group itself. Second, acetate and acetal hydrolysis were performed in one single step after hydrogenolysis.

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. Their capacity to inhibit aldolase catalyzed reaction by forming stabilized iminium ion or conjugated enamine were investigated by enzymatic kinetics and UV-vis difference spectroscopy. Depending of the substituent R (methyl or aromatic ring), a competitive or a slow-binding inhibition takes place. These results were examined on the basis of the three-dimensional structure of the enzyme.

The conserved residue Asp477 in yeast transketolase is located in the substrate channel of the enzyme and forms a hydrogen bond with the C2-hydroxyl group of the acceptor substrate. The significance of this interaction for the recognition of the preferred acceptor substrates, D-alpha-hydroxyaldehydes was investigated by site-directed mutagenesis. In the wild-type enzyme the kcat/KM values are by three to four orders of magnitude lower for 2-deoxyaldoses or substrates with L-configuration at the C2-atom. In the Asp477 Ala mutant, the kcat/KM values for D-alpha-hydroxyaldehydes are decreased by a thousandfold, while the kcat/KM values for substrates with L-configuration or 2-deoxyaldoses are similar to wild-type enzyme. These results indicate that Asp477 is involved in determining the enantioselectivity of transketolase.