Thermomonospora curvata is a thermophilic actinomycete hylogenetically related to Thermobifida fusca that produces extracellular hydrolases capable of degrading synthetic polyesters. Analysis of the genome of T. curvata DSM43183 revealed two genes coding for putative polyester hydrolases Tcur1278 and Tcur0390 sharing 61% sequence identity with the T. fusca enzymes. Mature proteins of Tcur1278 and Tcur0390 were cloned and expressed in Escherichia coli TOP10. Tcur1278 and Tcur0390 exhibited an optimal reaction temperature against p-nitrophenyl butyrate at 60°C and 55°C, respectively. The optimal pH for both enzymes was determined at pH 8.5. Tcur1278 retained more than 80% and Tcur0390 less than 10% of their initial activity following incubation for 60 min at 55°C. Tcur0390 showed a higher hydrolytic activity against poly(ε-caprolactone) and polyethylene terephthalate (PET) nanoparticles compared to Tcur1278 at reaction temperatures up to 50°C. At 55°C and 60°C, hydrolytic activity against PET nanoparticles was only detected with Tcur1278. In silico modeling of the polyester hydrolases and docking with a model substrate composed of two repeating units of PET revealed the typical fold of α/β serine hydrolases with an exposed catalytic triad. Molecular dynamics simulations confirmed the superior thermal stability of Tcur1278 considered as the main reason for its higher hydrolytic activity on PET.:Introduction; Materials and methods; Results; Discussion

Cytochrom P450 (CYP) Enzyme tragen zur Bioaktivierung von langkettigen mehrfach ungesättigten Fettsäuren bei. Die gebildeten Monoepoxy- und Monohydroxy-Metaboliten werden zusammenfassend als CYP-Eicosanoide bezeichnet und fungieren als Mediatoren bei der Regulation des Gefäßtonus, der Herz- und Nierenfunktion, sowie einer Vielzahl weiterer physiologischer Prozesse, wobei die biologische Aktivität oftmals abhängig von der Positions- und Stereoisomerie der Eicosanoide ist. Prominente Vertreter der CYP-Eicosanoid-Familie sind die aus der Arachidonsäure gebildeten Epoxyeicosatriensäuren (EETs) und 20-Hydroxyeicosatetraensäure (20-HETE). EETs und 20-HETE haben zum Teil gegensätzliche biologische Aktivitäten, die zur Aktivierung bzw. Inhibition antiinflammatorischer und weiterer Zell- und Organ-protektiver Signalwege beitragen. Ziel der vorliegenden Arbeit war es, durch Analyse endogener Metabolitenprofile zum besseren Verständnis der Rolle von CYP-Eicosanoiden bei der Entstehung von Ischämie/Reperfusions (I/R)-bedingten Organschäden beizutragen. Als Hauptergebnisse ergaben sich (i) die Entdeckung und Charakterisierung einer protektiven Rolle von EETs in Tiermodellen der Initiationsphase des akuten Nierenversagens, sowie des therapeutischen Potentials stabiler EET-Analoga; (ii) die Identifizierung von 8,9-EET und 20-HETE als mögliche prädiktive Biomarker für das post-operative Auftreten von akutem Nierenversagen nach offener Herzoperation; und (iii) die Entwicklung und Validierung eines analytischen Verfahrens der chiralen Lipidomik (chiral-LC-ESI-MS/MS), das eine Analyse endogener Enantiomere sowohl von Monoepoxy- als auch Monohydroxy-Eicosanoiden in komplexen biologischen Proben erstmalig ermöglichte und dafür genutzt werden konnte, die stereospezifische Regulation der EETs durch Epoxid-Hydrolasen in vitro wie auch in vivo zu beschreiben. / Cytochrome P450 (CYP) enzymes contribute to the bioactivation of long-chain polyunsaturated fatty acids. The monoepoxy- and monohydroxy-metabolites generated by CYP enzymes are collectively termed CYP-eicosanoids. CYP-eicosanoids act as mediators in the regulation of vascular tone, heart- and kidney function and several further physiological processes, mostly in a regio- and stereospecific manner. Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) are prominent members of the CYP-eicosanoid family. EETs and 20-HETE show partially opposing biological activities that contribute to the activation or inhibition of anti-inflammatory and other cell- and organ-protective mechanisms. The aim of the present work was to study the role of CYP-eicosanoids in ischemia/reperfusion (I/R) related organ damage by analyses of endogenous metabolite profiles. The main results were (i) discovery and characterization of the EETs protective role in animal models of the initiation phase of acute kidney injury (AKI) and the therapeutic potential of stable EET-analogs, (ii) identification of 8,9-EET and 20-HETE as potential predictive biomarkers for AKI in patients who underwent open heart surgery, (iii) the development and validation of a novel analytical method for chiral lipidomics (chiral LC-ESI-MS/MS) that allows to study endogenous enantiomers of monohydroxy- and monoepoxy-eicosanoids in complex matrices of biological and clinical samples. Furthermore, the approach was applied to describe the stereospecific regulation of EETs by epoxide hydrolases in vitro and in vivo.

Epoxide hydrolase I from Solanum tuberosum (StEH1) and isolated variants thereof has been studied for mapping structure-function relationships with the ultimate goal of being able to in silico predict modifications needed for a certain activity or selectivity. To solve this, directed evoultion using CASTing and an ISM approach was applied to improve selectivity towards either of the enantiomeric product diols from (2,3-epoxypropyl)benzene (1). A set of variants showing a range of activites and selectivities was isolated and characterized to show that both enantio- and regioselectivity was changed thus the enrichment in product purity was not solely due to kinetic resolution but also enantioconvergence. Chosen library residues do also influence selectivity and activity for other structurally similar epoxides styrene oxide (2), trans-2-methyl styrene oxide (3) and trans-stilbene oxide (5), despite these not being selected for.    The isolated hits were used to study varying selectivity and activity with different epoxides. The complex kinetic behaviour observed was combined with X-ray crystallization and QM/MM studies, powerful tools in trying to explain structure-function relationships. Crystal structures were solved for all isolated variants adding accuracy to the EVB calculations and the theoretical models did successfully reproduce experimental data for activities and selectivities in most cases for 2 and 5.  Major findings from calculations were that regioselectivity is not always determined in the alkylation step and for smaller and more flexible epoxides additional binding modes are possible, complicating predictions and the reaction scheme further. Involved residues for the catalytic mechanism were confirmed and a highly conserved histidine was found to have major influence on activity thus suggesting an expansion of the catalytic triad to also include H104. Docking of 1 into the active site of the solved crystal structures was performed in an attempt to rationalize regioselectivity from binding. This was indeed successful and an additional binding mode was identified, involving F33 and F189, both residues targeted for engineering. For biocatalytic purpose the enzyme were was successfully immobilized on alumina oxide membranes to function in a two-step biocatalytic reaction with immobilized alcoholdehydrogenase A from Rhodococcus ruber, producing 2-hydroxyacetophenone from racemic 2.

La peptidyl-ARNt hydrolase est une enzyme qui hydrolyse les peptidyl-ARNt issus d'une terminaison prématurée de la traduction. Cette protéine est essentielle à la viabilité des bactéries, mais pas à celle des eucaryotes, ce qui fait d'elle une cible potentielle pour l'action d'anti-bactériens. Cela justifie également qu'on cherche à cartographier l'interaction de cette protéine avec son substrat, pour faciliter la conception d'inhibiteur. Les tentatives d'obtention de cristaux de complexes enzyme:analogue de substrat étant restées vaines, nous avons choisi d'étudier de tels complexes en solution, par RMN. Grâce à un double marquage 15N/13C, nous avons tout d'abord attribué les fréquences de résonance des atomes du squelette de la protéine et d'une grande partie des chaînes latérale. Nous avons ensuite étudié l'interaction entre la PTH d'E. coli et un analogue de son substrat synthétisé chimiquement, la diacétyl-Lys-(3'NH)-adénosine. Cette étude nous a permis de caractériser le rôle de nombreux résidus du site actif, notamment celui d'une phénylalanine (F66) interagissant via son cycle aromatique avec l'adénine 3'-terminale du substrat, celui d'une asparagine (N114) stabilisant une molécule d'eau responsable de l'hydrolyse du substrat et celui d'une autre asparagine (N10) permettant à la PTH de discriminer positivement les peptidyl-ARNt par rapport aux aminoacyl-ARNt. Nous avons aussi étudié l'interaction entre la protéine et des mini-ARNt mimant la tige acceptrice et le bras TΨC d'un ARNt. Ce travail a permis de cartographier la surface de la PTH où l'ARN s'ancre à la protéine. Il a confirmé l'importance de deux résidus basiques, la lysine K105 et l'arginine R133, pour la reconnaissance du phosphate en 5' de l'ARNt. Il a également révélé une interaction entre l'hélice C-terminale de la protéine et le bras TΨC de l'ARNt, à 30 Å du site actif. La pertinence fonctionnelle de ce dernier contact a pu être établie par mutagenèse dirigée. L'ensemble de ces résultats permet de proposer un modèle complet de l'interaction entre la PTH et un peptidyl-ARNt.

<p>Molecular modelling strategies for evaluation of enzyme selectivity wereinvestigated with a focus on principles of how molecular interactionscould be evaluated to provide information about selectivity. Althoughmolecular modelling provides tools for evaluation of geometrical andenergy features of molecular systems, no general strategies for evaluationof enzyme selectivity exist. Geometrical analyses can be based uponinspection and reasoning about molecular interactions, which provide aneasily accessible way to gain information, but suffer from the risk of biasput in by the modeller. They can also be based on geometrical features ofmolecular interactions such as bond lengths and hydrogen-bond formation.Energy analyses are appealing for their modeller independenceand for the possibility to predict not only stereopreference, but also itsmagnitude.In this thesis, four examples of enantio- or regioselective serinehydrolase-catalysed reaction systems are presented together with developedmodelling protocols for explanation, prediction or enhancement ofselectivity. Geometrical as well as energy-based methodology were used,and provided an understanding of the structural basis of enzymeselectivity. In total, the protocols were successful in making qualitative explanationsand predictions of stereoselectivity, although quantitative determinationswere not achieved.</p>

No description available.

Agarose and porphyran are related galactans that are only found within red marine algae. As such, marine microorganisms have adapted to using these polysaccharides as carbon sources through the acquisition of unique Carbohydrate Active enZymes (CAZymes). A recent metagenome study of the microbiomes from a Japanese human population identified putative CAZymes in several bacterial species, including Bacteroides plebeius that have significant amino acid sequence similarity with those from marine bacteria. Analysis of one potential CAZyme from B. plebeius (BpGH50) is described here. While displaying up to 30% sequence identity with β-agarases, BpGH50 has no detectable agarase activity. Its crystal structure reveals that the topology of the active site is much different than previously characterized agarases, while containing the same core catalytic machinery. It is unclear whether the enzyme has endo- or exo- activity; the large binding ‘groove’ is typical of an endo-acting enzyme, while a loop at one end of the groove may provide a terminal pocket for the substrate, which is suggestive of exo-activity. Furthermore, the enzyme contains a basic pocket that may dock a sulphated substrate, like porphyran. While no quantifiable porphyran activity was observed, properties of the putative active site suggest that this unusual enzyme may be specific on an unusual substrate, such as a porphyran-agarose hybrid. / Graduate

The pyrimidine salvage enzyme, nucleoside hydrolase, is catalyzes the irreversible hydrolysis of nucleosides into the free nucleic acid base and D-ribose. Nucleoside hydrolases have varying degrees of specificity towards purine and pyrimidine nucleosides. In E. coli, three genes were found that encode homologues of several known nucleoside hydrolases in protozoa. All three genes (designated yaaF, yeiK, and ybeK) were amplified by PCR and cloned. Two of the gene products (yeiK and ybeK) encode pyrimidine-specific nucleoside hydrolases, while the third (yaaF) encodes a nonspecific nucleoside hydrolase. All three were expressed at low levels and had different modes of regulation. As a comparative analysis, the homologous genes of Pseudomonas aeruginosa and P. fluorescens (designated nuh) were cloned. Both were determined to encode nonspecific nucleoside hydrolases. The nucleoside hydrolases of the pseudomonads exhibited markedly different modes of regulation. Both have unique promoter structures and genetic organization. Furthermore, both pseudomonad nucleoside hydrolase were found to contain an N-terminal extension of 30-35 amino acids that is shown to act as a periplasmic-signaling sequence. These are the first two nucleoside hydrolases, to date,that have been conclusively demonstrated to be exported to the periplasmic space. The physiological relevance of this is explained.

La libération de l'acide arachidonique (AA) de la membrane des cellules par les phospholipases activées, le rend accessible pour être métabolisé par plusieurs enzymes impliquées dans la biosynthèse des eicosanoïdes. Ceux-ci incluent les cyclooxygénases (COX), les lipoxygénases (LOX) et plusieurs isoformes des cytochromes P450 (CYP450) qui produisent les acides époxy-eicosatriénoïques (EET) et les acides hydroxy-eicosatétraénoïques (HETE). Tandis qu'une attention considérable a été accordée aux rôles des eicosanoïdes dérivés des COX et des LOX, relativement peu de données sont disponibles sur les rôles potentiels des eicosanoïdes produits par les CYP450 sur la réactivité des muscles lisses des voies respiratoires (MLVR). L'objectif général de ce projet était de déterminer les modes d'action de ces eicosanoïdes bioactifs sur les tissus pulmonaires humains. L' idée étant de mieux cerner les mécanismes cellulaires et moléculaires activés lorsque ces tissus sont traités avec différents médiateurs lipidiques (eicosanoïdes) et inflammatoires (cytokines). Pour cela les propriétés électrophysiologiques, pharmacomécaniques et biochimiques susceptibles d'être modulées par ces eicosanoïdes sur les MLVR ont été analysées. Premièrement, nous avons démontré que l'acide 14,15-époxy-eicosatriénoïques (14,15-EET) hyperpolarise et relaxe les MLVR humains via l'activation des canaux potassiques de grande conductance activé par le Ca[exposant]2+ (BK[indice]Ca ). De plus, nous avons démontré que cet époxy-eicosanoïde diminue la sensibilité au Ca[indice]2+ des bronchioles perméabilisées à la [bêta]-escine, ce qui est corrélé avec une baisse du niveau de phosphorylation et d'expression de la protéine CPI-17 (protein kinase C-potentiated myosin phosphatase inhibitor). Par la suite, nous avons mis au point un modèle d'hyperréactivité bronchique (HRB) induit par un prétraitement des tissus au tumor necrosis factor [alpha] (TNF[alpha]). Ce modèle nous a permis d'évaluer les effets anti-inflammatoires des époxy-eicosanoïdes. Nous avons démontré que ces eicosanoïdes interfèrent avec l'activation du Nuclear Factor [kappa] B (NF[kappa]B) via leur interaction avec le peroxisome proliferator-activated receptors [gamma] (PPAR[gamma]). De plus, le 14,15-EET et l'acide 17,18-époxy-eicosatetraénoïque (17,18-EpETE) diminuent l'hypersensibilité au Ca[exposant]2+ des bronchioles prétraitées au TNF[alpha], en interférant avec la voie de signalisation de p38 mitogen-activated protein kinase (p38-MAPK), ce qui entraîne une réduction de la phosphorylation et de l'expression de la protéine CPI-17. L'introduction de siRNA dirigés contre les transcrits de la CPI-17 dans les bronchioles humaines traitées au TNF[alpha] a permis de mettre en évidence le rôle crucial de cette protéine dans l'HRB. Cette étude a aussi permis de démontrer que l'époxyde hydrolase soluble (sEH) est surexprimée dans les bronchioles humaines traitées au TNF[alpha] et dans les biopsies de patients asthmatiques. L'utilisation d'un inhibiteur pharmacologique de la sEH, le 12-(3-adamantyl-ureido)-dodecanoic acid (AUDA), a permis d'augmenter la biodisponibilité des époxy-eicosanoïdes et leurs effets bénéfiques sur les tissus bronchiques traités au TNF[alpha]. Les stratégies expérimentales établies ont permis de définir les modes d'action de ces eicosanoïdes et de démontrer leurs rôles potentiels contre l'HRB.

Inhibition of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the chief degradative enzymes of N-arachidonoyl ethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG), respectively, elicits no or partial substitution for Δ9-tetrahydrocannabinol (THC) in drug discrimination procedures. However, combined inhibition of both enzymes fully substitutes for THC, as well as produces a full constellation of cannabimimetic effects. Because no published report to date have investigated whether an inhibitor of endocannabinoid hydrolysis will serve as a discriminative stimulus, the purpose of this doctoral dissertation was to investigate whether C57BL/6J mice would learn to discriminate SA-57 (4-[2-(4-Chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester), a dual inhibitor of FAAH and MAGL, from vehicle in the drug discrimination paradigm. Also, we sought to determine whether inhibiting both enzymes, or inhibiting one enzyme was necessary to generate the SA-57 discriminative stimulus. Initial experiments showed that SA-57 fully substituted for either CP 55,940 ((-)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol), a high efficacy CB1 receptor agonist in C57BL/6J, mice or AEA in FAAH (-/-) mice. The majority (i.e., 23 of 24) of subjects achieved criteria of discriminating SA-57 (10 mg/kg) from vehicle within 40 sessions, with full generalization occurring 1-2 h post injection. CP 55,940, the dual FAAH-MAGL inhibitor JZL195 (4-nitrophenyl 4-(3-phenoxybenzyl)piperazine-1-carboxylate), the MAGL inhibitors MJN110 (2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate) and JZL184 (4-[Bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester) fully substituted for SA-57. Although, the FAAH inhibitors PF-3845 and URB597 did not substitute for SA-57, PF3845 produced a two-fold leftward shift in the MJN110 substitution dose-response curve. In addition, the CB1 receptor antagonist rimonabant blocked the generalization of SA-57 as well as substitution of CP 55,940, JZL195, MJN110, JZL184 for the SA-57 discriminative stimulus. These findings taken together indicate that the inhibition of endocannabinoid-regulating enzymes serve as breaks to prevent overstimulation of CB1 receptors, and MAGL inhibition is the major driving force for generating the SA-57 discriminative stimulus.