Etude de mécanismes moléculaires de l’évolution

Les travaux initiaux du laboratoire étaient orientés vers la description des mécanismes de l’adaptation de protéines isolées des Archaea halophiles. En analysant un grand nombre de données biochimiques, structurales au regard du concept de l’évolution, j’ai pu établir l’existence d’une nouvelle famille enzymatique : les LDH-like Malate Déshydrogénase dont je suis devenu un référent :(http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=cd01339).Toutes ces données ont été croisées à une étude phylogénétique qui m’a permis de décrire l’histoire évolutive de la super famille des malates (MalDH) et des lactates déshydrogénases (LDH). Cette famille est désormais considérée comme un vivier extrêmement riche pour trouver de nombreuses enzymes présentant des caractéristiques spécifiques d’adaptation à diverses contraintes physico-chimiques. Le dossier permet de voir que la combinaison de diverses méthodologies appliquées à l’étude de différentes enzymes (MalDH et LDH) permet de décortiquer en grands détails les mécanismes de l’adaptation. Les résultats obtenus font que cette famille enzymatique est une de celles qui est actuellement la mieux comprise en terme de relation repliement-structure-fonction-dynamique et évolution.Dans une deuxième partie, j’exposerai brièvement, comment ma réflexion a permis d’aller vers la caractérisation d’une nouvelle famille de déshydrogénase qui présente un nouveau motif de repliement, différent des déshydrogénases connues jusqu’à présent. Je montrerai aussi qu’il est vraisemblablement possible d’aller sonder les propriétés structurales d’une protéine ayant échappé au contrôle du repliemen

Asparaginyl-tRNA synthetase from the Escherichia coli temperature-sensitive strain HO202 A proline replacement in motif 2 is responsible for a large increase in Km for asparagine and ATP

AbstractThe Escherichia coli K12 mutant gene, asnS40, coding for asparaginyl-tRNA synthetase (AsnRS) in the temperature-sensitive strain HO202, was isolated from genomic DNA using the Polymerase Chain Reaction. DNA sequencing revealed that the mutant enzyme differs from the wild-type AsnRS by two amino acids, but only the P231L replacement leads to a change in aminoacylation activity. In the ATP-PPi exchange reaction at 37°C the purified P231L enzyme has a more than 50-fold increased Km value for asparagine compared to the wild-type enzyme, while the Km value for ATP is increased 8-fold. In the aminoacylation reaction the mutant enzyme shows also significantly increased Km values for asparagine and ATP. Interestingly Pro-231 is part of the conserved motif 2 in class II aminoacyl-tRNA synthetases (Eriani, G., Delarue, M., Poch, O., Gangloff, J. and Moras, D. (1990) Nature 347, 203–206), indicating that this motif might be involved in all class II enzymes in amino acid activation

Sampling the conformational energy landscape of a hyperthermophilic protein by engineering key substitutions

Proteins exist as a dynamic ensemble of interconverting substates, which defines their conformational energy landscapes. Recent work has indicated that mutations that shift the balance between conformational substates (CSs) are one of the main mechanisms by which proteins evolve new functions. In the present study, we probe this assertion by examining phenotypic protein adaptation to extreme conditions, using the allosteric tetrameric lactate dehydrogenase (LDH) from the hyperthermophilic bacterium Thermus thermophilus (Tt) as a model enzyme. In the presence of fructose 1, 6 bis-phosphate (FBP), allosteric LDHs catalyze the conversion of pyruvate to lactate with concomitant oxidation of nicotinamide adenine dinucleotide, reduced form (NADH). The catalysis involves a structural transition between a low-affinity inactive 'T-state' and a high-affinity active 'R-state' with bound FBP. During this structural transition, two important residues undergo changes in their side chain conformations. These are R171 and H188, which are involved in substrate and FBP binding, respectively. We designed two mutants of Tt-LDH with one ('1-Mut') and five ('5-Mut') mutations distant from the active site and characterized their catalytic, dynamical, and structural properties. In 1-Mut Tt-LDH, without FBP, the KmPyr is reduced compared with that of the wild type, which is consistent with a complete shifting of the CS equilibrium of H188 to that observed in the R-state. By contrast, the CS populations of R171, kcat and protein stability are little changed. In 5-Mut Tt-LDH, without FBP, KmPyr approaches the values it has with FBP and becomes almost temperature independent, kcat increases substantially, and the CS populations of R171 shift toward those of the R-state. These changes are accompanied by a decrease in protein stability at higher temperature, which is consistent with an increased flexibility at lower temperature. Together, these results show that the thermal properties of an enzyme can be strongly modified by only a few or even a single mutation, which serve to alter the equilibrium and, hence, the relative populations of functionally important native-state CSs, without changing the nature of the CSs themselves. They also provide insights into the types of mutational pathways by which protein adaptation to temperature is achieved.</p

ETUDES DES RELATIONS STRUCTURE-FONCTION, DU REPLIEMENT ET DE L'EVOLUTION AU SEIN DE LA SUPER-FAMILLE DES MALATES ET LACTATES DESHYDROGENASES

GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Phylogenetics and biochemistry elucidate the evolutionary link between l-malate and l-lactate dehydrogenases and disclose an intermediate group of sequences with mix functional properties

International audienceThe NAD(P)-dependent malate dehydrogenases (MDH) (EC 1.1.1.37) and NAD-dependent lactate dehydrogenases (LDH) (EC. 1.1.1.27) form a large super-family that has been characterized in organisms belonging to the three Domains of Life. MDH catalyses the reversible conversion of the oxaloacetate into malate, while LDH operates at the late stage of glycolysis by converting pyruvate into lactate. Phylogenetic studies proposed that the LDH/MDH superfamily encompasses five main groups of enzymes. Here, starting from 16,052 reference proteomes, we reinvestigated the relationship between MDH and LDH. We showed that the LDH/MDH superfamily encompasses three main families: MDH1, MDH2, and a large family encompassing MDH3, LDH, and L-2-hydroxyisocaproate dehydrogenases (HicDH) sequences. An in-depth analysis of the phylogeny of the MDH3/LDH/HicDH family and of the nature of three important amino acids located within the catalytic site and involved in binding and substrate discrimination, revealed a large group of sequences displaying unexpected combinations of amino acids at these three critical positions. This group branched in-between MDH3 and LDH sequences. The functional characterization of several enzymes from this intermediate group disclosed a mix of functional properties, indicating that the MDH3/LDH/HicDH family is much more diverse than previously thought, and blurred the frontier between MDH3 and LDH enzymes. Present-days enzymes of the intermediate group are a valuable material to study the evolutionary steps that led to functional diversity and emergence of allosteric regulation within the LDH/MDH superfamily

Protein thermal stability

International audienc

Stability against Denaturation Mechanisms in Halophilic Malate Dehydrogenase "Adapt" to Solvent Conditions

International audienceMalate dehydrogenase from Haloarcula marismortui (hMDH) is active, soluble and mildly unstable in an unusually wide range of salt conditions and temperatures, making it a particularly interesting model for the study of solvent effects on protein stability; Its denaturation (loss of activity due to concomitant dissociation and unfolding) kinetics was studied as a function of temperature and concentration of NaCl, potassium phosphate or ammonium sulphate in H2O or (H2O)-H-2. A transition-state-theory analysis was applied to the data. In all cases, stability (resistance to denaturation) increased with increasing salt concentration, and when (H2O)-H-2 replaced H2O. Each salt condition was associated with a particular energy regime that dominated stability. In NaCl/H2O, a positive enthalpy term, Delta H-not equal 0 , always dominated the activation free energy of denaturation, Delta G(not equal 0) . In potassium phosphate/H2O and ammonium sulphate/H2O, on the other hand, stability was dominated by a negative activation entropy Delta S-not equal 0, and Delta H-not equal 0 changed sign between 10 degrees C and 20 degrees C, consistent With a strong hydrophobic effect contribution, in these salting-out solvents. Decreasing stability at low temperatures, favouring cold denaturation, was observed. Replacing H2O by (H2O)-H-2 strengthened the hydrophobic effect in all conditions. As a consequence, conditions were found in which hMDH was not halophilic; below 10 degrees C, it was stable in approximate to 0.1 M NaCl/(H2O)-H-2. The solution structure and preferential solvent interactions of hMDH in H2O or (H2O)-H-2 solvents containing NaCl were studied by densimetry and neutron scattering. Despite the different stability of the protein in H2O or (H2O)-H-2, an experimentally identical invariant solution particle was formed in both solvents. It had a total volume of 1.165 cm(3) g(-1) and bound about 0.4 g of H2O (0.44 g of (H2O)-H-2) and about 0.08 g NaCl g protein. The impact of these results on a stabilisation model for hMDH, involving ion binding, is discussed

Intracellular molecular dynamics studied by neutron scattering

Incoherent neutron scattering experiments have produced important insights into intracellular molecular dynamics in vivo. Selected results highlight the role of water dynamics in cancer and brain cells, as well as cellular adaptation through the evolution of appropriate molecular dynamics, in order to respond to environmental challenges