Physiological factors that might influence ecological dynamics were investigated to better explain the biology of Bemisia tabaci in the desert southwest. Trehalulose, a unique disaccharide only found in unusually large quantities in B. tabaci honeydew, was shown not to be different from sucrose in promoting longevity in honeydew-consuming parasitoids, indicating this insect-modified sugar does not affect the nutritional quality of aleyrodid excreta. Trehalulose is not believed to function as a feeding deterrent to natural enemies. Experiments were designed to examine the effect of temperature on sorbitol and trehalulose production by the whitefly. High performance liquid chromatography analysis of honeydew and whole body extractions revealed a negative relationship between amounts of trehalulose in the honeydew and sorbitol accumulation in the whitefly body, linking these two molecules as important to the nutritional ecology of whiteflies. In another experiment to better understand the dispersal of whiteflies across the landscape, studies of flight over multiple days were conducted to describe the role of prior flight experience on dispersal and migratory flight. Flight performance traits were measured over multiple days of flight to compare two groups of B. tabaci, those trapped moving outside of planted fields with those collected within fields. Trap-caught individuals exhibited flights of significantly shorter duration in a vertical flight chamber. Flights determined to have characteristics of migratory behaviors were initially of longer duration for trap-captured whiteflies than their field-collected counterparts. Over the context of multiple days, however, their longer flights were followed by much shorter flights on subsequent days. Although many insects from both groups were capable of movement on multiple days, almost all of these flights were of a foraging nature. Foraging flights of short duration would likely not add to dispersal distances, thereby limiting whiteflies to their originating patch.

Les amylosaccharases sont des α-transglucosylases catalysant naturellement la synthèse exclusive d’α-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 α-allyl-N-acetyl-2-désoxy-α-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 catalytique / Amylosucrases are sucrose-utilizing α-transglucosylases that naturally catalyze the synthesis of α-glucans, exclusively linked through α-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 α-ally-N-acetyl-2-deoxy-α-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 process