Recent years have seen tremendous developments in methods for computational modeling of (bio-) molecular systems. Ever larger reactive systems are being studied with high accuracy approaches, and high-level QM/MM calculations are being routinely performed. However, applying high-accuracy methods to large biological systems is computationally expensive and becomes problematic when conformational sampling is needed. To address this challenge, classical force field based approaches such as free energy perturbation (FEP) and empirical valence bond calculations (EVB) have been employed in this work. Specifically: Force-field independent metal parameters have been developed for a range of alkaline earth and transition metal ions, which successfully reproduce experimental solvation free energies, metal-oxygen distances, and coordination numbers. These are valuable for the computational study of biological systems. Experimental studies have shown that the epoxide hydrolase from Solanum tuberosum (StEH1) is not only an enantioselective enzyme, but for smaller substrates, displays enantioconvergent behavior. For StEH1, two detailed studies, involving combined experimental and computational efforts have been performed: We first used trans-stilbene oxide to establish the basic reaction mechanism of this enzyme. Importantly, a highly conserved and earlier ignored histidine was identified to be important for catalysis. Following from this, EVB and experiment have been used to investigate the enantioconvergence of the StEH1-catalyzed hydrolysis of styrene oxide. This combined approach involved wildtype StEH1 and an engineered enzyme variant, and established a molecular understanding of enantioconvergent behavior of StEH1. A novel framework was developed for the Computer-Aided Directed Evolution of Enzymes (CADEE), in order to be able to quickly prepare, simulate, and analyze hundreds of enzyme variants. CADEE’s easy applicability is demonstrated in the form of an educational example. In conclusion, classical approaches are a computationally economical means to achieve extensive conformational sampling. Using the EVB approach has enabled me to obtain a molecular understanding of complex enzymatic systems. I have also increased the reach of the EVB approach, through the implementation of CADEE, which enables efficient and highly parallel in silico testing of hundreds-to-thousands of individual enzyme variants.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第22400号 / 薬科博第122号 / 新制||薬科||13(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 掛谷 秀昭, 教授 二木 史朗, 教授 土居 雅夫 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

No tubo digestivo das larvas de Anastrepha fraterculus e Anastrepha pickeli há β-glicosidases capazes de clivar dissacarideos, β-glicosídeos tóxicos produzidos por plantas e substratos sintéticos. As β-glicosidases de A. fraterculus são pouco ativas e as de A. pickeli são bastante ativas sobre alguns compostos, entre eles linamarina, um glicosídeo cianogênico. Esse composto está presente, em altas concentrações, no fruto da mandioca do qual a larva se alimenta. A. fraterculus alimenta-se do fruto da goiaba e aparentemente consegue o carboidrato que necessita por ação de α-glicosidases, que são bem mais ativas do que as β. O fruto da mandioca não é tão nutritivo e A. pickeli deve aproveitar a glicose da linamarina para obter energia e consegue desintoxicar-se do aglicone tóxico. Rhynchosciara americana apresenta quatro β-glicosidases nas membranas microvilares intestinais, sendo três delas β-galactosidases. Dessas, duas são ativadas por Triton X-100 sendo que a glicosidase, de maior mobilidade eletroforética é ativada por este composto, com uma Ka de 4µM, um α de 0,5 e um β de 2. β-tioglicosidases foram demonstradas em afideos. Nós verificamos que ocorre a clivagem do tioglicosídeo sinigrina após separação das β-glicosidases digestivas do Lepidoptera Diatraea saccharalis por cromatografia hidrofóbica. Nesse inseto, a mesma enzima é capaz de clivar O- e S-glicosídeos com atividades semelhantes. Enzimas com essas características nunca foram descritas anteriormente. Esses experimentos ilustram a viabilidade das adaptações dos insetos na utilização de compostos formados for ligações β-glicosídicas, viabilizando a exploração de nutrientes normalmente inacessíveis a outros animais. / Anastrepha fraterculus and Anastrepha pickeli have in their midguts 13-glycosidases able to hydrolase dissaccharides, synthetic substrate and plant toxic β-glucosides. β-glycosidases from A. fraterculus have low activity and the enzymes from A. pickeli may be highly active depending on the substrate used. Linamarin, a cyanogenic β-glucoside present in A. pickeli food (Manihot fruit) is easly hydrolysed by A. pickeli β-glycosidases (A. fraterculus eats on guava fruits and may obtain carbohydrate through the action of α-glycosidases, that are much more active them the β-glycosidases). A. pickeli probably uses glucose derived from linamarinan avoiding the effects of the toxic aglycon. Rhynchosciara americana has 4 β-glycosidases (3 galactosidases and I glucosidase) in their intestinal microvilar membranes. Two of these enzymes are activated by Triton X-100. In β glucosidase the activation has Ka= 4µM, α=0,5 e β=2. β-thioglycosidases occur in Aphids. One digestive β-glucosidase from Diatraea saccharalis resolved by hydrofobic chrornatography hydrolyses sinigrin. The same enzyme may hydrolyse O- and S-glucosides with the same efficienly. Enzymes with this specificity have never been described before. In this study we shown some adaptations of insects to use substrates with β-glycosidic bonds, allowing these organisrns to explore nutrients usualy avoided by other animals.

A importância do estudo das celulases não se limita à aquisição de conhecimento científico, mas também ao grande potencial biotecnológico que elas representam. Isso se deve ao fato da celulose ser a molécula mais abundante presente na natureza e prover uma vasta gama de produtos e processos sustentáveis. Muitas famílias de celulases já foram bem caracterizadas, enquanto outras permanecem ainda desconhecidas. Dentre estas últimas, a família 45 das hidrolases de glicosídeos é a família de celulases fúngicas menos caracterizada tanto estruturalmente quanto funcionalmente. Recentemente foi proposta a divisão dessa família em três subfamílias e, até agora, apenas membros da subfamília A tiveram enzimas estruturalmente elucidadas. Nesse trabalho reportamos a estrutura cristalográfica da proteína recombinante endoglucanase de Phanerochaete chrysosporium (PcCel45A), a primeira das hidrolases de glicosídeos da subfamília C, e seu complexo com celobiose a 1,4 Å e 1,7 Å de resolução, respectivamente. A PcCel45A é uma enzima de domínio único, com uma estrutura em β-barril e seu empacotamento geral remete ao formato de âncora. O sítio ativo da enzima forma um longo sulco na superfície da estrutura, sendo que o seu centro catalítico é diferente das outras enzimas publicadas dessa família e o aspartato catalítico, que atua como aceptor de próton na reação de inversão, (Asp10) não é conservado. Adicionalmente, a estrutura cristalográfica dessa enzima apresenta mais similaridades com as β-expansinas (proteínas de plantas) e transglicosilases líticas (proteínas que clivam o peptidoglicano de bactérias) do que com as outras representantes da família 45, o que a torna ainda mais singular. Para entendermos melhor seu funcionamento foram realizadas mutações sítio-dirigidas nos principais resíduos do sítio ativo. O Asp121, conhecido por participar da reação de inversão das outras enzimas da família como doador de próton, mostrou-se essencial para a atividade da enzima, enquanto que outros resíduos conservados como a Tyr25, o Trp161 e o Asp92 afetaram, mas não aniquilaram a atividade da enzima, apresentando aproximadamente 20%, 50% e 10% da atividade da enzima nativa, respectivamente. / The importance of the study of the cellulases is not limited to generating significant scientific knowledge, since these enzymes represents an enormous potential in biotechnology. This is partly because cellulose is the most abundant molecule in nature and provides a wide range of products and sustainable process. Many cellulases families have been well characterized, while others still remain unknown. Among them, the glycoside hydrolase family 45 is the least well characterized both structurally and functionally, between fungal cellulases. It was recently proposed the subdivision of this family into three subfamilies, with structural information available only for subfamily A. In this work, we report the chrystallographic structure of the recombinant endoglucanase from Phanerochaete chrysosporium (PcCel45A), the first GH45 subfamily C and its complex with cellobiose at 1.4 Å and 1.7 Å respectively. The PcCel45A is a single domain enzyme, which has a β-barrel structure with the overall shape resembling an anchor. The active site of the enzyme has a long cleft on the surface, being remarkably different from those members of subfamily A, and the catalytic aspartate responsible for acting as proton acceptor (Asp10) is not present. Additionally, the chrystallographic structure of this enzyme has shown more similarity with β -expansins (plant proteins) and lytic transglycosylase (proteins that cleave the peptidoglycan of bacteria) than others representants of family 45, which makes it more singular. For a better understanding of its function, we perform pontual mutations in the main residues from active site. The Asp121, known for acting as proton acceptor in the inversion reaction of others enzymes, proved to be essential for the enzyme activity, while others conserved residues as Tyr25, Trp161 and Asp92 affected but not annihilated the enzyme activity, leaving approximately 20%, 50% and 10% of the native enzyme activity.

Multiple sclerosis (MS) is a demyelinating neurodegenerative disease that typically has a relapsing-remitting pattern of progression superimposed on a gradual worsening of disease symptoms. Experimental autoimmune encephalomyelitis (EAE) is a model of MS where animals develop relapses, demyelination and accumulate neurological deficits. Studies using the EAE model have provided evidence that cannabinoids are beneficial in reducing disease symptoms and may impact long term neurodegeneration, but side-effects of exogenous cannabinoid receptor agonists may limit their potential as therapeutic agents for MS. Targeting enzymes involved in degradation of endocannabinoids such as the anandamide-degrading enzyme fatty acid amide hydrolase (FAAH) may be an attractive alternative strategy. Using experimental allergic encephalomyelitis (EAE) as a mouse model of MS, two complementary approaches were used to assess FAAH as a potential therapeutic target. The FAAH deficient (ABH.FAAH-/-) developed similar paralytic relapsing disease of similar severity of disease compared to the wild-type, but showed a poorer recovery following the acute phase. However, following a relapsing-remitting disease course, the FAAH deficient mice showed a substantial improvement in clinical score, improved motor control, and lost less neurofilament compared to wild-type mice. These findings indicate that fatty acid amides may be neuroprotective in EAE. Secondly, a selective FAAH inhibitor (PF-3845; 10 mg/kg) was used to treat mice during the relapse phase of the disease course. Treatment with PF-3845 caused an elevation of anandamide in the CNS. This treatment resulted in a small reduction in neurofilament loss, but no reduction in clinical score or improvement in motor control was observed compared to the vehicle treated group. To investigate at a cellular level how FAAH might affect disease progression in the EAE model, immunohistochemistry was used to analyse FAAH expression in the CNS. Employing novel antibodies to FAAH in combination with neuronal and glial cell markers, it was found that, in addition to previously reported neuronal expression of FAAH, FAAH is highly expressed 3 in oligodendrocytes, but not in other glial cell types. Thus, genetic deletion or pharmacological inhibition of FAAH may affect both neuronal activity and oligodendroglial function (e.g. myelination). The role of FAAH in oligodendrocytes was investigated in vitro. An oligodendrocyte precursor cell (OPC) monoculture was used to monitor differentiation, and a co-culture comprising neurons and OPCs was used to monitor myelination. During the differentiation of OPCs, FAAH expression was detected in the entire oligodendroglia lineage, but with high expression only in mature myelin basic protein (MBP) expressing cells. Treatment with the FAAH inhibitor PF-3845 (0.1 μM to 1 μM) increased differentiation of OPCs into mature oligodendrocytes. However, the same treatment of co-cultures had no effect on the myelination of neurites. In conclusion, this study has: i) obtained evidence that genetic deletion of FAAH is neuroprotective in a mouse model of MS and ii) provided new insights on FAAH expression in the CNS. Further investigation of FAAH, in particular its role(s) in oligodendrocytes, will be required to fully unlock the therapeutic potential of FAAH inhibition in the treatment of MS.

Epóxido hidrolases (EHs) são enzimas que catalisam a hidrólise de epóxidos a seus correspondentes dióis, apresentam potencial aplicação biotecnológica (separação de enantiômeros na produção de fármacos), estão envolvidas no metabolismo de ácidos graxos poliinsaturados e inibidores de EHs estão sendo estudados para possível utilização no tratamento de doenças. Uma enzima epóxido hidrolase (TrEH) do fungo filamentoso Trichoderma reesei QM9414 foi clonada, expressa, purificada e caracterizada funcionalmente e estruturalmente. A atividade de TrEH foi determinada com o substrato óxido de estireno (racêmico), demonstrando maior atividade nas temperaturas de 23 a 50 °C, no pH 7,2 a 37 °C, e as constantes cataliticas Km= 4,6 mM e kcat= 336 s-1. A enzima recombinante mostrou ser enantiosseletiva, pois hidrolisa preferencialmente (S)-(-)-óxido de estireno, (R)-(-)- epicloridrina e (S)-(-)-1,2-epoxibutano. Moléculas inibidoras da atividade de TrEH foram identificadas e algumas delas inibem até 60% o crescimento de T. reesei. A estrutura terciária de TrEH (1,7 Å) foi determinada por cristalografia, apresenta dobramento α/β-hidrolase e não tem alta homologia com nenhuma outra estrutura de EH. TrEH é uma nova enzima epóxido hidrolase solúvel cujas propriedades mostram seu potencial de utilização em aplicações biotecnológicas. / Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to their corresponding diols, present a potential biotechnological application (separation of enantiomers for the production of drugs), they are involved in the metabolism of polyunsaturated fatty acids and EH inhibitors are being studied for possible use in the treatment of diseases. An epoxide hydrolase enzyme(TrEH) from the filamentous fungus Trichoderma reesei QM9414 was cloned, expressed, purified and functionally and structurally characterized. The activity of TrEH was determined with the substrate styrene oxide (racemic), showing higher activity at temperatures of 23 to 50 °C, at pH 7.2 at 37 °C, and the catalytic constants Km= 4.6 mM and kcat= 336 s-1. The recombinant enzyme has been shown to be enantioselective, because it preferably hydrolyzes (S)-(-)-styrene oxide, (R)-(-)-epichlorohydrin and (S)-(-)-1,2- epoxybutane. TrEH inhibitors have been identified and some of them inhibit up to 60% growth of T. reesei . The tertiary structure of TrEH (1.7 Å) was determined by crystallography, showing α/ β-hydrolase folding and low homology with any other EH structure. TrEH is a new soluble epoxide hydrolase enzyme whose properties show its potential for use in biotechnological applications.

N-acylethanolamines (NAEs) are derived from a minor membrane lipid constituent N-acylphosphatidylethanolamine and are hydrolyzed by fatty acid amide hydrolases (FAAH) into free fatty acid (FFA) and ethanolamine in both plants and animals. In Arabidopsis, NAE plays an important physiological role in growth/development and response to stress. Although NAEs are reported in tomato, their metabolic pathway remains undiscovered. It is hypothesized that there is a functional FAAH in tomato that hydrolyzes NAEs. To this extent, a putative gene that likely encodes for putative SlFAAH1 protein was identified, cloned, and heterologously expressed. Amidase activity was tested using radiolabeled NAE substrates. Furthermore, expression of putative SlFAAH1 transcripts and protein activity was quantified at different developmental stages to demonstrate endogenous amidase activity in tomato seedlings. In future, molecular and biochemical characterization of tomato FAAH will further test the conserved nature of NAE metabolic pathway in plants.

Overdose and addiction are two medical complications of cocaine abuse. To date, there is no FDA approved pharmacotherapy specific for cocaine abuse. Cocaine hydrolases (CocHs) have been extensively investigated for its potential in anti-cocaine therapy. Previous studies have demonstrated that CocHs efficiently hydrolyze cocaine to generate biologically inactive metabolites both in vivo and in vitro. However, it has not been studied whether there is gender difference in the therapy using CocHs. In addition, the effectiveness of CocHs is unknown for treating cocaine toxicity when alcohol is co-administered. The main purpose of this dissertation is to characterize and evaluate efficient CocHs for cocaine overdose and cocaine addiction treatment. In the first set of studies, the effectiveness of human serum albumin-fused CocH1 were studied in male and female rats. The pharmacokinetic profiles, as well as the pharmacodynamic effects of CocH1-HSA were compared in male and female rats. The obtained data clearly demonstrated that CocH1-HSA was equally effective in both genders. The second set of studies investigated the efficiency of Fc-fused CocH5 in reversing cocaine toxicity in rats receiving simultaneous administration of cocaine and alcohol. Results showed that CocH5-Fc rapidly hydrolyzed cocaine and cocaine’s toxic metabolites in rats, and demonstrated that CocH5-Fc was efficient in treating cocaine toxicity when alcohol was simultaneously administered. In later studies to investigate the effects of CocH5-Fc for the treatment of cocaine addiction, a mathematical model was developed and validated to predict the effects of CocH5-Fc on the disposition of cocaine in rat blood and brain. This model adequately described the effects of CocH5-Fc in accelerating the elimination of cocaine and its toxic metabolites in both rat blood and brain. In conclusion, the studies within the current dissertation demonstrate the clinical potential of CocHs for the treatment of both cocaine overdose and cocaine addiction.

No description available.

Le peptidoglycane est un composant majeur de la paroi cellulaire bactérienne dont l'intégrité est essentielle à la survie cellulaire. La biosynthèse du peptidoglycane est un processus régulé faisant intervenir des enzymes de synthèse, les « Penicillin-Binding Proteins », et des hydrolases qui agissent, selon toute vraisemblance, de façon concertée lors de la croissance et de la division bactérienne. La comparaison des génomes bactériens a permis d'identifier un nouveau domaine protéique, probablement impliqué dans le clivage du peptidoglycane, présent uniquement chez les bactéries à Gram positif : le domaine PECACE (PEptidoglycan CArbohydrate Cleavage Enzyme). Ce domaine, prédit pour avoir un repliement tridimensionnel analogue aux domaines catalytiques de la transglycosylase lytique Slt70 d'Escherichia coli et du lysozyme de type G d'oie, pourrait participer au clivage de la liaison glycosidique β(1-4) entre les résidus acide N-acétyl-D-muramique et N-acétyl-D-glucosamine du peptidoglycane. Chez la bactérie pathogène Streptococcus pneumoniae, ce domaine est présent dans la région extracellulaire d'une protéine que nous nommons Pmp23 (Pneumococcal Membrane Protein). La forme recombinante de cette protéine membranaire bitopique de 23 kDa dégrade in vitro des extraits bactériens contenant du peptidoglycane. L'inactivation du gène pmp23 chez S. pneumoniae modifie le comportement de la bactérie en culture, accroît sa sensibilité aux antibiotiques de la famille des β-lactamines et perturbe la localisation du site de division. Ces observations indiquent que Pmp23 est une hydrolase intervenant dans le métabolisme du peptidoglycane septal.