Caractérisation structurale et fonctionnelle d'amylosaccharases

Les amylosaccharases sont des a-transglucosylases catalysant naturellement la synthèse exclusive d a-1,4-glucanes à partir du saccharose. Ces enzymes produisent également des composés secondaires et, en particulier, des isomères du saccharose tels que le turanose et le tréhalulose.L objectif de cette thèse a consisté à utiliser un panel de techniques biophysiques et biochimiques afin d étudier les amylosaccharases de Deinococcus geothermalis (ASDg) et Neisseria polysaccharea (ASNp) afin de comprendre les relations unissant la structure, la flexibilité et la fonction de ces enzymes.La première étude rapporte la caractérisation structurale et biophysique de l amylosaccharase la plus thermostable connue à ce jour, l amylosaccharase de Deinococcus geothermalis. La structure tridimensionnelle révèle une organisation dimérique en solution, jamais rapportée pour une amylosaccharase. Grâce à l analyse de l interface dimérique et à des travaux d analyse de séquences, une séquence signature de dimérisation a été identifiée. En rigidifiant la structure de l ASDg, la structure quaternaire contribue à l augmentation de la stabilité thermique de la protéine. La spécificité de production des isomères du saccharose par les amylosaccharases a été étudiée. Les résultats décrivent, pour la première fois, les structures de l ASDg et de l ASNp en complexe avec le turanose. Dans l ASNp, les résidus clefs forcent le résidu fructosyle à adopter une conformation linéaire positionnant idéalement le O3 pour sa glucosylation expliquant la formation préférentielle de turanose par l enzyme. Ces résidus sont absents ou placés différemment dans l ASDg. En conséquence, l ASDg lie principalement les formes furanoses du fructose avec un faible réseau d interactions. La topologie du sous-site +1 permet donc différents modes de liaison du fructose en accord avec la capacité de l ASDg à produire une plus grande quantité de tréhalulose par rapport à l ASNp.Dans la seconde étude, des techniques de mutagenèse à saturation et combinatoire ciblées sur les acides aminés voisins du site actif ont été utilisées pour modifier la spécificité d'accepteur de l ASNp. Le criblage de trois bibliothèques semi-rationnelles représentant un total de 20 000 variants a permis d isoler trois doubles mutants montrant une amélioration spectaculaire de spécificité à la fois vis-à-vis du saccharose, le substrat donneur et de l accepteur a-allyl-N-acetyl-2-désoxy-a-D-glucopyranoside par rapport au type sauvage de l ASNp. De tels niveaux d'amélioration d'activité n'ont jamais été signalés auparavant pour cette classe d enzymes actives sur les sucres. L analyse par cristallographie des rayons X de la structure des meilleures enzymes mutantes suivie par des simulations de dynamique moléculaire ont montré une rigidité locale du sous-site -1 couplée à une flexibilité des boucles impliquées dans la topologie du site actif. Ces faits pourraient être à l origine des performances catalytiques accrues de ces enzymes mutantes. L'étude démontre l'importance, lors de la conception des bibliothèques de variants, de tenir compte de la conformation locale des résidus catalytiques ainsi que de la dynamique des protéines au cours du processus catalytiqueAmylosucrases are sucrose-utilizing a-transglucosylases that naturally catalyze the synthesis of a-glucans, exclusively linked through a-1,4 linkages. Side-products and in particular sucrose isomers such as turanose and trehalulose are also produced by these enzymes.The objective of this thesis concerned the application of biophysical and biochemical techniques to study amylosucrases from Deinococcus geothermalis (DgAS) and Neisseria polysaccharea (NpAS) in order to investigate relationships between structure, flexibility and function of these enzymes.In the first study, we report the first structural and biophysical characterization of the most thermostable amylosucrase identified so far, the amylosucrase from Deinoccocus geothermalis. The 3D-structure revealed a homodimeric quaternary organization, never reported before for other amylosucrases. A sequence signature of dimerization was identified from the analysis of the dimer interface and sequence alignments. By rigidifying DgAS structure, the quaternary organization is likely to participate in the enhanced thermal stability of the protein. Amylosucrase specificity with respect to sucrose isomer formation (turanose or trehalulose) was also investigated. We report the first structures of the DgAS and NpAS in complex with turanose. In NpAS, key residues were found to force the fructosyl moiety to bind in an open state with the O3' ideally positioned to explain the preferential formation of turanose by NpAS. Such residues are either not present or not similarly placed in DgAS. As a consequence, DgAS binds the furanose tautomers of fructose through a weak network of interactions to enable turanose formation. Such topology at subsite +1 is likely favoring other possible fructose binding modes in agreement with the higher amount of trehalulose formed by DgAS.In the second study, iterative saturation mutagenesis and combinatorial active site saturation focused on vicinal amino acids were used to alter the acceptor specificity of NpAS and sort out improved variants. From the screening of three semi-rational sub-libraries accounting in total for 20,000 variants, we report here the isolation of three double-mutants displaying a spectacular specificity enhancement towards both sucrose, the donor substrate, and the a-ally-N-acetyl-2-deoxy-a-D-glucopyranoside acceptor compared to wild-type N. polysaccharea amylosucrase. Such levels of activity improvement have never been reported before for this class of carbohydrate-active enzymes. X-ray structural analysis of the best performing enzymes followed by Molecular Dynamics simulations showed both local rigidity of the -1 subsite and flexibility of loops involved in active site topology which both account for the enhanced catalytic performances of the mutants. The study well illustrates the importance when designing enzyme libraries of taking into account the local conformation of catalytic residues as well as protein dynamics during the catalytic processTOULOUSE-INSA-Bib. electronique (315559905) / SudocSudocFranceF

Similarities and differences in the biochemical and enzymological properties of the four isomaltases from Saccharomyces cerevisiae

AbstractThe yeast Saccharomyces cerevisiae IMA multigene family encodes four isomaltases sharing high sequence identity from 65% to 99%. Here, we explore their functional diversity, with exhaustive in-vitro characterization of their enzymological and biochemical properties. The four isoenzymes exhibited a preference for the α-(1,6) disaccharides isomaltose and palatinose, with Michaëlis–Menten kinetics and inhibition at high substrates concentration. They were also able to hydrolyze trisaccharides bearing an α-(1,6) linkage, but also α-(1,2), α-(1,3) and α-(1,5) disaccharides including sucrose, highlighting their substrate ambiguity. While Ima1p and Ima2p presented almost identical characteristics, our results nevertheless showed many singularities within this protein family. In particular, Ima3p presented lower activities and thermostability than Ima2p despite only three different amino acids between the sequences of these two isoforms. The Ima3p_R279Q variant recovered activity levels of Ima2p, while the Leu-to-Pro substitution at position 240 significantly increased the stability of Ima3p and supported the role of prolines in thermostability. The most distant protein, Ima5p, presented the lowest optimal temperature and was also extremely sensitive to temperature. Isomaltose hydrolysis by Ima5p challenged previous conclusions about the requirement of specific amino acids for determining the specificity for α-(1,6) substrates. We finally found a mixed inhibition by maltose for Ima5p while, contrary to a previous work, Ima1p inhibition by maltose was competitive at very low isomaltose concentrations and uncompetitive as the substrate concentration increased. Altogether, this work illustrates that a gene family encoding proteins with strong sequence similarities can lead to enzyme with notable differences in biochemical and enzymological properties

Distinction between Pore Assembly by Staphylococcal α-Toxin versus Leukotoxins

The staphylococcal bipartite leukotoxins and the homoheptameric α-toxin belong to the same family of β-barrel pore-forming toxins despite slight differences. In the α-toxin pore, the N-terminal extremity of each protomer interacts as a deployed latch with two consecutive protomers in the vicinity of the pore lumen. N-terminal extremities of leukotoxins as seen in their three-dimensional structures are heterogeneous in length and take part in the β-sandwich core of soluble monomers. Hence, the interaction of these N-terminal extremities within structures of adjacent monomers is questionable. We show here that modifications of their N-termini by two different processes, using fusion with glutathione S-transferase (GST) and bridging of the N-terminal extremity to the adjacent β-sheet via disulphide bridges, are not deleterious for biological activity. Therefore, bipartite leukotoxins do not need a large extension of their N-terminal extremities to form functional pores, thus illustrating a microheterogeneity of the structural organizations between bipartite leukotoxins and α-toxin

Etude en solution de la protéine TorD de Shewanella massilia et détermination de la structure tridimensionnelle du dimère, par la méthode MAD

En anaérobiose, certaines bactéries utilisent le triméthylamine N-oxyde (TMAO) comme accepteur exogène d'électron. Cette voie métabolique implique l'expression de l'opéron tor qui code notamment pour une TMAO réductase périplasmique (TorA), pour un cytochrome pentahémique de type c (TorC), et pour une chaperone cytoplasmique (TorD) requise pour l'acquisition du cofacteur à molybdène et la translocation de la réductase par le système tat. Dans ce travail, nous montrons que la chaperone TorD de Shewanella massilia présente plusieurs états oligomériques stables. Les formes monomérique, dimérique et trimérique ont été purifiées et caractérisées en solution. Les données de diffusion des rayons X aux petits angles et de diffraction indiquent que TorD dimère est constitué de deux domaines identiques et de taille similaire à celle du monomère. La structure cristallographique du dimère de TorD à 2,4 A de résolution révèle que la dimérisation s'effectue par échange de domaine au cours duquel la moitié N-terminale d'un protomère s'associe à la moitié C-terminale d'un autre et réciproquement. L'interconversion entre les différentes espèces se produit à pH acide. Dans ces conditions, la protéine est dans un état partiellement dénaturé contenant une proportion d'hélices alpha identique à celle des formes natives. Il a par ailleurs été montré que les formes monomérique et dimérique se lient à l'enzyme TorA mature, mais que le dimère présente une affinité supérieure. Le dimère de TorD présente des structures tertiaire et quaternaire originales. D'après les identités de séquences, ce nouveau repliement est probablement représentatif des chaperones associées aux DMSO/TMAO réductases bactériennes ainsi que d'autres protéines: identifiées dont la fonction est encore inconnue.Several bacteria use trimethylamine N-oxyde (TMAO) as an exogenous electron acceptor for anaerobic respiration. This metabolic pathway involves expression of the tor operon that codes for a periplasmic molybdopterin-containing reductase of the DMSO/TMAO family (TorA), a pentahemic c-type cytochrome (TorC) and the TorD cytoplasmic chaperone required for acquisition of the molybdenum cofactor and translocation of the reductase by the twin-arginine translocation system. In this work, we show that the TorD chaperone from Shewanella massilia forms multiple and stable oligomeric species. The monomeric, dimeric and trimeric forms were purified to homogenity and characterized in solution. Small-angle X-ray scattering (SAXS) and crystallographic data indicate that the TorD dimer is made of identical protein modules of similar size to the monomeric species. The X-ray structure at 2.4 A resolution of the TorD dimer reveals extreme domain swapping between the two subunits where the N-terminal part of a protomer interacts with the C-terminal part of the other protomer. Interconversion of the native oligomeric forms occurred at acidic pH value. In this condition, the protein is in a non native conformation containing a fraction of alpha-helices similar to that of the native species. It has also been shown that both the monomeric and the dimeric species bind the mature TorA enzyme, but that the dimer binds its target protein more efficiently. The TorD dimer shows no similarity with known protein structures. According to sequence similarities, this new fold probably represents the architecture of the chaperones associated to the bacterial DMSO/TMAO reductases and also that of identified proteins of Jet unknown functions.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Structural bases for N-glycan processing by mannoside phosphorylase

The first crystal structure of Uhgb_MP, a β-1,4-mannopyranosyl-chitobiose phosphorylase belonging to the GH130 family which is involved in N-glycan degradation by human gut bacteria, was solved at 1.85 Å resolution in the apo form and in complex with mannose and N-acetylglucosamine. SAXS and crystal structure analysis revealed a hexameric structure, a specific feature of GH130 enzymes among other glycoside phosphorylases. Mapping of the -1 and +1 subsites in the presence of phosphate confirmed the conserved Asp104 as the general acid/base catalytic residue, which is in agreement with a single-step reaction mechanism involving Man O3 assistance for proton transfer. Analysis of this structure, the first to be solved for a member of the GH130_2 subfamily, revealed Met67, Phe203 and the Gly121-Pro125 loop as the main determinants of the specificity of Uhgb_MP and its homologues towards the N-glycan core oligosaccharides and mannan, and the molecular bases of the key role played by GH130 enzymes in the catabolism of dietary fibre and host glycans

Structural Insights into the Carbohydrate Binding Ability of an α-(1→2) Branching Sucrase from Glycoside Hydrolase Family 70

International audienceThe alpha-(1 -> 2) branching sucrase Delta N-123-GBD-CD2 is a transglucosylase belonging to glycoside hydrolase family 70 (GH70) that catalyzes the transfer of D-glucosyl units from sucrose to dextrans or gluco-oligosaccharides via the formation of alpha-(1 -> 2) glucosidic linkages. The first structures of Delta N-123-GBD-CD2 in complex with D-glucose, isomaltosyl, or isomaltotriosyl residues were solved. The glucose complex revealed three glucose-binding sites in the catalytic gorge and six additional binding sites at the surface of domains B, IV, and V. Soaking with isomaltotriose or gluco-oligosaccharides led to structures in which isomaltosyl or isomaltotriosyl residues were found in glucan binding pockets located in domain V. One aromatic residue is systematically identified at the bottom of these pockets in stacking interaction with one glucosyl moiety. The carbohydrate is also maintained by a network of hydrogen bonds and van der Waals interactions. The sequence of these binding pockets is conserved and repeatedly present in domain V of several GH70 glucansucrases known to bind alpha-glucans. These findings provide the first structural evidence of the molecular interaction occurring between isomalto-oligosaccharides and domain V of the GH70 enzymes

Characterizationof a muli-modular GH3-CBM48-CE1 isolated for termite gut microbiota

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Neutral Genetic Drift-Based Engineering of a Sucrose-Utilizing Enzyme toward Glycodiversification

Neutral drift (also called purifying selection) is an attractive approach to generate polymorphic variant libraries for enzyme engineering. Here, we have applied this strategy to modify the substrate specificity of a transglucosylase. Our model enzyme, the amylosucrase from Neisseria polysaccharea, is a glucosylationbiocatalyst of prime interest because it uses the widespread substrate sucrose as a glucosyl donor and shows broad acceptor promiscuity. A library of 440 functional amylosucrase variants was generated after four rounds of neutral drift at a low mutation rate. The functional variations present in this library were investigated byassaying the ability of these variants to use an alternative glucosyl donor (pnitrophenyl-α-D-glucopyranoside, pNP-Glc) and to glucosylate a range of acceptors (including methyl-α-L-rhamnopyranoside, which is not naturally recognized by the parental enzyme). The impact of these mutations on the thermal stability of thevariants was also assessed. Large variations of acceptor promiscuity were observed, ranging from the complete loss of detectable activity to a 2-fold increase relative to the parental enzyme. Variants showing increased catalytic efficiency toward the alternative pNP-Glc donor were also identified. Specifically, one variant combining four unprecedented amino acid changes was 25-fold more efficient at utilizing pNP-Glc than the parental enzyme and acquired glucosylation activity toward methyl-α-L-rhamnopyranoside. Enzymes with improved thermalstability were also identified. Overall, our work demonstrates that neutral drift is an effective and powerful strategy to engineer transglycosylases with enhanced or even acquired substrate specificities from small-sized functional libraries compatible with accurate low-throughput multi-parameter analyses

Engineering of anp efficient mutant of Neisseria polysaccharea amylosucrase for the synthesis of controlled size maltooligosaccharides

Amylosucrase from Neisseria polysaccharea naturally catalyzes the synthesis of α-1,4 glucans from sucrose. The product profile is quite polydisperse, ranging from soluble chains called maltooligosaccharides to high-molecular weight insoluble amylose. This enzyme was recently subjected to engineering of its active site to enable recognition of non-natural acceptor substrates. Libraries of variants were constructed and screened on sucrose, allowing the identification of a mutant that showed a 6-fold enhanced activity toward sucrose compared to the wild-type enzyme. Furthermore, its product profile was unprecedented, as only soluble maltooligosaccharides of controlled size chains (2 < DP < 21) with a narrow polydispersity were observed. This variant, containing 9 mutations in the active site, was characterized at both biochemical and structural levels. Its x-ray structure was determined and further investigated by molecular dynamics to understand the molecular origins of its higher activity on sucrose and higher production of small maltooligosaccharides, with a totally abolished insoluble glucan synthesis