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1	Characterization of Metal-binding P-loop GTPases: E. coli YeiR and M. thermoacetica AcsF Flood, Jessica 23 November 2011 (has links) Organisms express metal-binding proteins in order to deal with essential metal ions that can be potentially toxic. A common trend of bacterial metal homeostasis pathways is the presence of a GTPase, and several of these proteins are members of the G3E family of P-loop GTPases. In this work we focused on E. coli YeiR, member of the under-characterized COG0523 subfamily, and on M. thermoacetica AcsF. The in vitro metal-binding properties of isolated YeiR are presented. The protein binds Ni2+ and Zn2+ with low micromolar affinity, and oligomerizes in the presence of metal. The GTPase activity of YeiR is similar to that measured for other members of the group and is enhanced by Zn2+. In the case of AcsF, it was not possible to establish concluding evidence that the protein binds metal. This study helps shed light upon members of the under-characterized subfamily of G3E P-loop GTPases. P-loop GTPase metallochaperone 0487
2	Characterization of Metal-binding P-loop GTPases: E. coli YeiR and M. thermoacetica AcsF Flood, Jessica 23 November 2011 (has links) Organisms express metal-binding proteins in order to deal with essential metal ions that can be potentially toxic. A common trend of bacterial metal homeostasis pathways is the presence of a GTPase, and several of these proteins are members of the G3E family of P-loop GTPases. In this work we focused on E. coli YeiR, member of the under-characterized COG0523 subfamily, and on M. thermoacetica AcsF. The in vitro metal-binding properties of isolated YeiR are presented. The protein binds Ni2+ and Zn2+ with low micromolar affinity, and oligomerizes in the presence of metal. The GTPase activity of YeiR is similar to that measured for other members of the group and is enhanced by Zn2+. In the case of AcsF, it was not possible to establish concluding evidence that the protein binds metal. This study helps shed light upon members of the under-characterized subfamily of G3E P-loop GTPases. P-loop GTPase metallochaperone 0487
3	Identification et caractérisation d'une enzyme bifonctionnelle de Ruminococcus gnavus E1 (AgaSK), présentant une activité [alpha]-galactosidase et une activité kinase / Identification and characterization of a bifunctional enzyme from Ruminococcus gnavus E1 (AgaSK) coupling galactosidase and kinase activities Bruel, Laëtitia 25 March 2014 (has links) Les α-galactosides sont des glucides non digestibles constitués d'unités galactose liées en α(1,6). Les α-galactosides de la famille du raffinose (RFO) sont, avec le saccharose, les principaux oligosaccharides des légumineuses. Cependant, aucune activité α(1,6)-galactosidase n'est retrouvée au niveau de l'épithélium intestinal humain, les RFO sont donc exclusivement fermentés par les enzymes microbiennes. Ces travaux introduisent une enzyme bifonctionnelle de Ruminococcus gnavus E1, un membre majoritaire du microbiote intestinal humain, présentant une activité α(1,6)-galactosidase/ saccharose kinase (AgaSK). L'analyse de la séquence peptidique montre qu'AgaSK présente deux domaines : un domaine homologue aux α-galactosidases GH36, et un autre contenant un motif de fixation des nucléotides (motif A de Walker). La caractérisation des paramètres biochimiques d'AgaSK met en évidence cette bifonctionnalité puisqu'elle est capable d'hydrolyser les α(1,6)-galactosides solubles, et parallèlement en présence d'ATP de phosphoryler le saccharose spécifiquement sur la position C6 du glucose. La production directe de saccharose-6-phosphate à partir de l'hydrolyse du raffinose constitue une voie métabolique jamais décrite chez les bactéries. L'analyse de chacun des domaines montre que les domaines isolés d'AgaSK sont actifs mais la comparaison de leurs paramètres cinétiques montre qu'il y a des différences entre la protéine entière et les domaines isolés. La résolution de la structure du domaine α-galactosidase en complexe avec le galactose démontre que l'état oligomérique est nécessaire pour le bon repliement de la protéine et pour une fixation efficace du substrat. / Α-galactosides are non digestible carbohydrates present in many leguminous plants. Soluble α-galactosides consist of galactose units α(1,6) linked to different carbohydrates. Among these, the raffinose family oligosaccharides (RFO) and sucrose, are the most abundant oligosaccharides found in legumes. However, no α(1,6)galactosidase activity exists in the human intestine mucosa and α-galactosides are exclusively fermented by microbial α(1,6)galactosidases (EC3.2.1.22). Here we introduce a bifunctional enzyme, the α(1,6)galactosidase/sucrose kinase (AgaSK) whose gene is highly transcribed in vivo by Ruminococcus gnavus E1, a major member of human dominant intestinal microbiota. Sequence analysis showed that AgaSK is composed of two domains: one closely related to α-galactosidases from glycoside hydrolase family GH36 and the other containing a nucleotide binding motif (Walker A motif). Its biochemical characterization showed that AgaSK is able to hydrolyze efficiently soluble α-galacosides. Furthermore, AgaSK it is able to bind nucleotide to phosphorylate specifically on the C6 position of glucose sucrose. The production of sucrose-6-P directly from raffinose brings out a glycolytic pathway in bacteria, not described so far. In addition, AgaSK isolated domains are active but the biochemical characterization has shown that there are differences in the activities between the whole protein and isolated domains. The crystal structures of the galactosidase domain in complex with the product shed light onto the reaction and substrate recognition mechanisms and highlight an oligomeric state necessary for efficient substrate binding. Saccharose Enzyme bifonctionnelle [Αlpha]-Galactosidase Saccharose kinase P-Loop Microbiote intestinal humain Raffinose family oligosaccarides Sucrose Bifunctionnal enzyme [Αlpha]-Galactosidase Sucrose kinase P-Loop Human gut microbiota
4	Electrostaticanalisys the Ras active site Khan, Abdul Kareem 05 March 2009 (has links) La preorganització electrostàtica del centre actiu s'ha postulat com el mecanisme genèric de l'acció dels enzims. Així, alguns residus "estratègics" es disposarien per catalitzar reaccions interaccionant en una forma més forta amb l'estat de transició, baixant d'aquesta manera el valor de l'energia dactivació g cat. S'ha proposat que aquesta preorientació electrostática s'hauria de poder mostrar analitzant l'estabilitat electrostàtica de residus individuals en el centre actiu.Ras es una proteïna essencial de senyalització i actúa com un interruptor cel.lular. Les característiques estructurals de Ras en el seu estat actiu (ON) són diferents de les que té a l'estat inactiu (OFF). En aquesta tesi es duu a terme una anàlisi exhaustiva de l'estabilitat dels residus del centre actiu deRas en l'estat actiu i inactiu. / The electrostatic preorganization of the active site has been put forward as the general framework of action of enzymes. Thus, enzymes would position "strategic" residues in such a way to be prepared to catalyze reactions byinteracting in a stronger way with the transition state, in this way decreasing the activation energy g cat for the catalytic process. It has been proposed thatsuch electrostatic preorientation should be shown by analyzing the electrostatic stability of individual residues in the active site.Ras protein is an essential signaling molecule and functions as a switch in thecell. The structural features of the Ras protein in its active state (ON state) are different than those in its inactive state (OFF state). In this thesis, an exhaustive analysis of the stability of residues in the active and inactive Ras active site is performed. dinàmica centre actiu relacions d'estructura activitat preorganització reorganització estat de transició estat actiu estabilitat electrostàtica interacción proteïna-proteïna test de ranks de Wilcoxon P-loop G1 motif HVR metastability RMSD substrate assisted catalysis sequence alignment beta sheet helix Z-test wilcoxon rank test protein-protein interactions electrostatic stability active state dynamics signal transduction electrostatic stabilization ras effectors guanine exchange factor GTPase-activating proteins quantum mechanics/molecular PDLD/S-LRA X-ray crystallography solvation energy free energy perturbation statistical analysis GTP hydrolysis guanosine diphosphate guanosine triphosphate computer simulation thermodynamics protein conformation electrostatics interruptor II interruptor I llaç P motiu G1 HVR (regions altament variables) metaestabilita RMSD catàlisi assistida pel substrat aliniament de seqüència fulla beta hèlix test Z transducció del senyal estabilització electrostàtica efectors de ras factor de bescamvi de guanina proteïnes activadores de l'activitat mecànica quàntica/mecànica PDLD/S-LRA cristalografia de raigs X energia de solvatació perturbació de l'energia lliure anàlisi estadística hidròlisi de GTP difosfat de guanosina trifosfat de guanosina simulació computacional termodinàmica conformació proteïca electrostàtica switch-I switch-II 537 576

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