Rates of Chemical Reactions Embedded in a Metabolic Network by Dissolution Dynamic Nuclear Polarisation NMR

International audienceThe isomerisation of 6-phosphogluconolactones and their hydrolyses into 6-phosphogluconic acid form a non enzymatic side cycle of the pentose-phosphate pathway (PPP) in cells. We show that dissolution dynamic nuclear polarization can be used for determining the kinetic rates of the involved transformations in real time. It is found that the hydrolysis of both lactones is significantly slower than the isomeration process, thereby shedding new light onto this subtle chemical process

Etudes de cinétiques enzymatiques par polarisation dynamique nucléaire avec dissolution (D-DNP) : application à l'étape oxydative de la voie des pentoses phosphates (PPP)

The Pentose Phosphate Pathway (PPP) is one of the main pathways of cellular metabolism. This metabolic pathway is composed of two enzymatic cascades: one is an oxidative pathway, and the other is non-oxidative. The oxidative branch of PPP produces a cofactor, NADPH, which is responsible for the detoxification process of the cell due to its reducing activity, and is also a precursor of various biosynthesis such as lipogenesis. A dysfunction of one of the three enzymes that make up this step of PPP can lead to cell death. Thanks to a new method, Dissolution Dynamic Nuclear Polarization (D-DNP), which features a sensitivity gain by a factor 10,000 compared to standard liquid-state NMR, the quantification of kinetic parameters under physiological conditions, in cell, becomes possible.In this thesis, we add to the scientific library a new model of quantification of kinetic parameters, and the possibility of studying an enzymatic cascade composed of 3 enzymes by D-DNP measurements. Based on these experiments, the selectivity of the first enzyme in the oxidative pathway, G6PD, for one of the two glucose-6-phosphate anomers, was confirmed. The antioxidant role of the second PPP enzyme, 6PGL, was equally studied. To carry out these studies, a method of synthesis and purification of the different substrates of each enzyme has been developed. The very first inhibitor of 6PGL has also been tested. Preliminary experiments on Trypanosoma brucei, a parasite responsible for sleeping sickness, indicate that glucose penetration inside cells is the limiting kinetic step for its conversion.L'une des voies principales du métabolisme cellulaire est la voie des Pentoses Phosphates (PPP). Cette voie métabolique est composée de deux cascades enzymatiques, une voie oxydative et une voie non oxydative. La voie oxydative de la PPP produit un cofacteur, le NADPH, qui est responsable du processus de détoxification de la cellule par son activité réductrice et un précurseur de diverses biosynthèses comme la lipogenèse. Un dysfonctionnement des trois enzymes qui composent cette étape de la PPP peut engendrer la mort cellulaire. Grâce à une nouvelle technique, la Polarisation Dynamique Nucléaire suivie par Dissolution (D-DNP), qui permet d’obtenir un gain de sensibilité par un facteur > 10 000, la quantification des paramètres cinétique dans les conditions physiologiques in cell est possible.Dans ce travail de thèse, nous utilisons un nouveau modèle de quantification des paramètres cinétiques qui offre la possibilité d’étudier une cascade enzymatique composée de 3 enzymes par D-DNP. Grâce à ces expériences, la sélectivité de la première enzyme de la voie oxydative, la G6PD, pour l’un des deux anomères de glucose-6-phosphate, ainsi que le rôle antioxydant de la deuxième enzyme de la PPP, la 6PGL, ont été observés. Pour réaliser ces études, une méthode de synthèse et de purification des différents substrats de chaque enzyme a été développée. Le tout premier inhibiteur de la 6PGL a été testé. Des études préliminaires réalisées sur des Trypanosoma brucei, parasite responsable de la maladie du sommeil, indiquent que la pénétration du glucose dans les cellules est l'étape cinétiquement limitante pour sa conversion enzymatique

Enzymatic kinetic studies by nuclear dynamic polarization with dissolution (D-DNP) : application to the oxidative step of the pentose phosphate pathway (PPP)

L'une des voies principales du métabolisme cellulaire est la voie des Pentoses Phosphates (PPP). Cette voie métabolique est composée de deux cascades enzymatiques, une voie oxydative et une voie non oxydative. La voie oxydative de la PPP produit un cofacteur, le NADPH, qui est responsable du processus de détoxification de la cellule par son activité réductrice et un précurseur de diverses biosynthèses comme la lipogenèse. Un dysfonctionnement des trois enzymes qui composent cette étape de la PPP peut engendrer la mort cellulaire. Grâce à une nouvelle technique, la Polarisation Dynamique Nucléaire suivie par Dissolution (D-DNP), qui permet d’obtenir un gain de sensibilité par un facteur > 10 000, la quantification des paramètres cinétique dans les conditions physiologiques in cell est possible.Dans ce travail de thèse, nous utilisons un nouveau modèle de quantification des paramètres cinétiques qui offre la possibilité d’étudier une cascade enzymatique composée de 3 enzymes par D-DNP. Grâce à ces expériences, la sélectivité de la première enzyme de la voie oxydative, la G6PD, pour l’un des deux anomères de glucose-6-phosphate, ainsi que le rôle antioxydant de la deuxième enzyme de la PPP, la 6PGL, ont été observés. Pour réaliser ces études, une méthode de synthèse et de purification des différents substrats de chaque enzyme a été développée. Le tout premier inhibiteur de la 6PGL a été testé. Des études préliminaires réalisées sur des Trypanosoma brucei, parasite responsable de la maladie du sommeil, indiquent que la pénétration du glucose dans les cellules est l'étape cinétiquement limitante pour sa conversion enzymatique.The Pentose Phosphate Pathway (PPP) is one of the main pathways of cellular metabolism. This metabolic pathway is composed of two enzymatic cascades: one is an oxidative pathway, and the other is non-oxidative. The oxidative branch of PPP produces a cofactor, NADPH, which is responsible for the detoxification process of the cell due to its reducing activity, and is also a precursor of various biosynthesis such as lipogenesis. A dysfunction of one of the three enzymes that make up this step of PPP can lead to cell death. Thanks to a new method, Dissolution Dynamic Nuclear Polarization (D-DNP), which features a sensitivity gain by a factor 10,000 compared to standard liquid-state NMR, the quantification of kinetic parameters under physiological conditions, in cell, becomes possible.In this thesis, we add to the scientific library a new model of quantification of kinetic parameters, and the possibility of studying an enzymatic cascade composed of 3 enzymes by D-DNP measurements. Based on these experiments, the selectivity of the first enzyme in the oxidative pathway, G6PD, for one of the two glucose-6-phosphate anomers, was confirmed. The antioxidant role of the second PPP enzyme, 6PGL, was equally studied. To carry out these studies, a method of synthesis and purification of the different substrates of each enzyme has been developed. The very first inhibitor of 6PGL has also been tested. Preliminary experiments on Trypanosoma brucei, a parasite responsible for sleeping sickness, indicate that glucose penetration inside cells is the limiting kinetic step for its conversion

Rotating-Frame Overhauser Transfer via Long-Lived Coherences

Solution-state distance restraints for protein structure determination with Ångström-level resolution rely on through-space transfer of magnetization between nuclear spins. Such magnetization transfers, named Overhauser effects, occur via dipolar magnetic couplings. We demonstrate improvements in magnetization transfer using long-lived coherences (LLCs)—singlet-triplet superpositions that are antisymmetric with respect to spin-permutation within pairs of coupled magnetic nuclei—as the magnetization source. Magnetization transfers in the presence of radio-frequency irradiation, known as ‘rotating-frame’ Overhauser effects (ROEs), are predicted by theory to improve by the use of LLCs; calculations are matched by preliminary experiments herein. The LLC-ROE transfers were compared to the transmission of magnetization via classical transverse routes. Long-lived coherences accumulate magnetization on an external third proton, K, with transfer rates that depended on the tumbling regime. I,S →K transfers in the LLC configuration for (I,S) are anticipated to match, and then overcome, the same transfer rates in the classical configuration as the molecular rotational correlation times increase. Experimentally, we measured the LLC-ROE transfer in dipeptide AlaGly between aliphatic protons in different residues K = Ala − Hα and (I,S) = Gly − Hα1,2 over a distance dK,I,S = 2.3 Å. Based on spin dynamics calculations, we anticipate that, for such distances, a superior transfer of magnetization occurs using LLC-ROE compared to classical ROE at correlation times above τC=10 ns. The LLC-ROE effect shows potential for improving structural studies of large proteins and offering constraints of increased precision for high-affinity protein-ligand complexes in slow tumbling in the liquid state

Targeting the Pentose Phosphate Pathway: Characterization of a New 6PGL Inhibitor

International audienceHuman African trypanosomiasis, or sleeping sickness, is a lethal disease caused by the protozoan parasite Trypanosoma brucei. However, although many efforts have been made to understand the biochemistry of this parasite, drug development has led to treatments that are of limited efficiency and of great toxicity. To develop new drugs, new targets must be identified, and among the several metabolic processes of trypanosomes that have been proposed as drug targets, carbohydrate metabolism (glycolysis and the pentose phosphate pathway (PPP)) appears as a promising one. As far as the PPP is concerned, a limited number of studies are related to the glucose-6-phosphate dehydrogenase. In this work, we have focused on the activity of the second PPP enzyme (6-phospho-gluconolactonase (6PGL)) that transforms 6-phosphogluconolactone into 6-phosphogluconic acid. A lactam analog of the natural substrate has been synthesized, and binding of the ligand to 6PGL has been investigated by NMR titration. The ability of this ligand to inhibit 6PGL has also been demonstrated using ultraviolet experiments, and protein-inhibitor interactions have been investigated through docking calculations and molecular dynamics simulations. In addition, a marginal inhibition of the third enzyme of the PPP (6-phosphogluconate dehydrogenase) was also demonstrated. Our results thus open new prospects for targeting T. brucei

Structure and function of natural proteins for water transport: general discussion

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Dissolution dynamic nuclear polarization of deuterated molecules enhanced by cross-polarization

The authors want to thank Dr. Jean-Nicolas Dumez for helpful discussions; Sven Sieber, Michael Schenkel, Rolf Hensel, Jacco van Beek, Ion Prisucaru, and Armin Purea for contributions to the system design and implementation; and Frank Engelke, Pietro Lendi, Klemens Kessler, Daniel Eckert, Daniel Guy Baumann, Dirk Wilhelm, Roberto Seydoux, Uwe Wagner, Cengiz Cetrefli, Tonio Gianotti, and Jorg Hinderer for contributions to the system specification. Some of the work in this article employed the SpinDynamica code for Mathematica, programmed by Malcolm H. Levitt, with contributions from Jyrki Rantaharju, Andreas Brinkmann, and Soumya Singha Roy, available at www.SpinDynamica.soton.ac.uk.International audienceWe present novel means to hyperpolarize deuterium nuclei in 13CD2 groups at cryogenic temperatures. The method is based on cross-polarization from 1H to 13C and does not require any radio-frequency fields applied to the deuterium nuclei. After rapid dissolution, a new class of long-lived spin states can be detected indirectly by 13C NMR in solution. These long-lived states result from a sextet-triplet imbalance (STI) that involves the two equivalent deuterons with spin I = 1. An STI has similar properties as a triplet-singlet imbalance that can occur in systems with two equivalent I = 12/ spins. Although the lifetimes T STI are shorter than T 1(Cz), they can exceed the life-time T 1(Dz) of deuterium Zeeman magnetization by a factor of more than 20