Abstract NADH-ubiquinone oxidoreductase (complex I) is one of the largest enzymes in mammals. Seven (ND1-ND6 and ND4L) of its 45 subunits are encoded in mitochondrial DNA, mutations of which are usually behind mitochondrial diseases such as Leber hereditary optic neuropathy (LHON) and MELAS-syndrome. The rest of the genes are located in the nucleus. Bacterial homologs of complex I (NDH-1) consist of only 13–14 subunits, comprising the catalytic core of the enzyme. These complexes are simpler but perform a similar function. Escherichia coli NDH-1 was employed here to generate amino acid replacements at conserved sites in NuoH, NuoJ and NuoK, counterparts of ND1, ND6 and ND4L, to elucidate their role in complex I. Consequences of homologous amino acid substitutions brought about by ND1-affecting LHON/MELAS-overlap syndrome-associated m.3376G>A and m.3865A>G mutations and the ND6-affecting m.14498T>C substitution associated with LHON were also studied to validate their pathogenicity. Effects of the site-directed mutations were evaluated on the basis of enzyme activity, inhibitor sensitivity and growth phenotype. Highly conserved glutamate-residues 36 and 72 within transmembrane helices of NuoK in positions similar to proton translocating transmembrane proteins were found essential for electron transfer to ubiquinone and growth on medium necessitating normal proton transfer by NDH-1. NuoH and NuoJ replacements at sites corresponding to targets of m.3376G>A and m.14498T>C decreased ubiquinone reductase activity and altered the ubiquinone binding site, while the counterpart of m.3865A>G was without a major effect. Other NuoH and NuoJ mutations studied also affected the interactions of ubiquinone and inhibitors with NDH-1. The results corroborate the pathogenicity of the m.14498T>C and m.3376G>A mutations and demonstrate that the overlap syndrome-associated modification affects complex I in a pattern which appears to combine the effects of separate mutations responsible for LHON and MELAS. Change in ubiquinone binding affinity is a likely pathomechanism of all LHON-associated mutations. Effects of the NuoH, NuoJ and NuoK subunit substitutions also indicate that ND1 and ND6 subunits contribute to the ubiquinone-interacting site of complex I and the site is located in the vicinity of the membrane surface, while ND4L is likely involved in proton pumping activity of the enzyme. / Tiivistelmä 45 alayksiköstä muodostuva NADH-ubikinoni oksidoreduktaasi (kompleksi I) on nisäkkäiden suurimpia entsyymejä. Sen mitokondriaalisessa DNA:ssa koodattujen alayksiköiden ND1-ND6 ja ND4L geeneihin liittyvät mutaatiot ovat yleisiä mitokondriosairauksien, kuten Leberin perinnöllisen näköhermoatrofian (LHON) ja MELAS-oireyhtymän, syitä. Bakteerien vastaava entsyymi (NDH-1) koostuu vain 13–14 alayksiköstä. Tästä huolimatta sen katalysoima reaktio on samankaltainen kuin kompleksi I:n. NDH-1:n katsotaankin edustavan entsyymin katalyyttistä ydintä. Tässä työssä tutkittiin ND1, ND6 ja ND4L alayksiköiden tehtävää kompleksi I:ssä niiden Escherichia coli bakteerissa olevien vastineiden (NuoH, NuoJ ja NuoK) kohdennetun mutageneesin avulla. Samaa lähestymistapaa käytettiin LHON/MELAS-oireyhtymässä todettujen ND1 alayksikön mutaatioiden, m.3376G>A ja m.3865A>G, ja LHON:ssa havaitun ND6:n m.14498T>C mutaation aiheuttamien aminohappomuutosten seurauksien selvittämiseen. Tehtyjen mutaatioiden vaikutuksia arvioitiin entsyymiaktiivisuus-mittauksin ja kasvukokein. NuoK:n solukalvon läpäisevissä rakenteissa olevien kahden glutamaatti-aminohappotähteen sijainti muistuttaa protoneita kalvon läpi kuljettavissa proteiineissa todettua. NuoK:n glutamaattien havaittiinkin olevan tärkeitä elektronien ja protonien kuljetukselle kompleksi I:ssä. m.3376G>A ja m.14498T>C mutaatioiden aiheuttamien aminohappomuutosten vastineet NDH-1:ssä alensivat NDH-1:n elektroninsiirtoaktiivisuutta ja heikensivät ubikinonin sitoutumista, kun taas m.3865A>G mutaatiolla ei ollut vaikutusta. Muut NuoH ja NuoJ alayksiköihin tehdyt aminohappovaihdokset johtivat huonontuneeseen ubikinonin ja kompleksi I:n inhibiittoreiden sitoutumiseen. Saadut tulokset vahvistavat m.3376G>A ja m.14498T>C mutaatioiden patogeenisyyden. Ne myös osoittavat, että LHON/MELAS-oireyhtymään liitetyn mutaation biokemiallisissa vaikutuksissa yhdistyvät sekä LHON:ssa että MELAS-oireyhtymässä todettujen mutaatioiden seuraukset. Esitetyt tulokset tukevat näkemystä siitä, että ubikinonin ja kompleksi I:n välisessä vuorovaikutuksessa tapahtuva muutos on kaikille LHON:aan liitetyille mutaatioille yhteinen vaikutusmekanismi. NuoH:n, NuoJ:n ja NuoK:n kohdennetusta mutageneesista saatujen tulosten perusteella ND1 ja ND6 alayksiköt ovat osa ubikinonin sitoutumispaikkaa entsyymikompleksissa, kun taas ND4L osallistuu protoninkuljetukseen.

The thesis describes structural and functional studies of two PLP-dependent enzymes, diaminopropionate (DAP) ammonia lyase (DAPAL) and N-acetylornithine aminotransferase (AcOAT). The main objective of this work was to understand the structural features that control and impart specificity for PLP-dependent catalysis. DAPAL is a prokaryotic enzyme that catalyzes the degradation of D and L forms of DAP to pyruvate and ammonia. The first crystal structure of DAPAL was determined from Escherichia coli (EcDAPAL) in holo and apo forms, and in complex with various ligands. The structure with a transient reaction intermediate (aminoacrylate-PLP azomethine) bound at the active site was obtained from crystals soaked with substrate, DL-DAP. Apo and holo structures revealed that the region around the active site undergoes transition from disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. Based on the crystal structures and biochemical studies, as well as studies on active site mutant enzymes, a two base mechanism of catalysis involving Asp120 and Lys77 is suggested. AcOAT is an enzyme of arginine biosynthesis pathway that catalyses the reversible conversion of N-acetylglutamate semialdehyde and glutamate to N-acetyl ornithine and α-ketoglutarate. It belongs to subgroup III of fold type I PLP dependent enzymes. Many clinically important aminotransferases belong to the same subgroup and share many structural similarities. We have carried out extensive comparative analysis of these enzymes to identify the unique features important for substrate specificity. Crystal structures of AcOAT from Salmonella typhimurium were determined in presence of two ligands, canaline and gabaculine, which are known to act as general inhibitors for most of the enzymes of this class. There structures provided important insights into the mode of binding of the substrates. The structures illustrated the switching of conformation of an active site glutamate side chain on binding of the two substrates. In addition to that, structural transitions involving three loop regions near the active site were observed in different ligand bound structures. Kinetics of single turnover fast reactions and multiple turnover steady state reactions indicated that N-AcOAT dimer might follow a mechanism involving sequential half site reactivity for efficient catalysis. The changes observed in loop conformation that resulted in asymmetric forms of the dimer enzyme might form the structural basis for half site reactivity. Single site mutants were designed to understand the significance of these structural transitions and the specific role of active site residues in determining substrate specificity and catalysis. Biochemical characterization of wild type and mutant enzymes by steady state and fast kinetic studies, along with their crystal structures provided detailed insights into subtlety of active site features that manifest substrate specificity and catalytic activity. The thesis also describes the investigations on fold type II enzymes directed towards analyses of polypeptide folds of these enzymes, features of their active sites, nature of interactions between the cofactor and the polypeptide, oligomeric structure, catalytic activities with various ligands, origin of specificity and plausible regulation of activity. Analysis of the available crystal structures of fold type II enzymes revealed five different classes. The dimeric interfaces found in these enzymes vary across the classes and probably have functional significance. Contributions made towards structural and functional studies of three other PLP-dependent enzymes, serine hydoxymethyltransferase (SHMT), D-serine deaminase (DSD) and D-cysteine desulfhydrase (DCyD) are described in an appendix.

Fibronectin is an extracellular multidomain glycoprotein that directs and regulates a variety of cell processes such as proliferation, development, haemostasis, embryogenesis, and wound healing. As a major component of blood, fibronectin exists as a soluble disulphide linked dimer, but it can also be incorporated into an insoluble cross-linked fibrillar network to form a major component of the extracellular matrix. Fibronectin is composed of an extended chain of module repeats termed Fn1, Fn2, and Fn3 that bind to a wide range of transmembrane receptors and extracellular matrix components, including collagen. The gelatin binding domain of fibronectin was first isolated as a 45kDa proteolytic fragment and has since been found to be composed of six modules: 6Fn1-1Fn2-2Fn2-7Fn1-8Fn1-9Fn1 (in this notation nFX represents the nth type X module in the native protein). This domain has been reported to bind to both collagen and denatured collagen (gelatin), but with 10-100 times higher affinity to the latter; it can be purified to homogeneity on a gelatin affinity column. In the work presented here, fragments of the gelatin binding domain are expressed in P. pastoris, purified to homogeneity, and investigated at the molecular level. Through a dissection approach, surface plasmon resonance (SPR) is used to characterise the recombinantly produced protein, to accumulate more information about the function of the full domain. NMR is used to assess the folding of the protein fragments at atomic resolution. In particular, the secondary structure of 8Fn1-9Fn1 is mapped using inter-strand NOEs, which suggests that the construct takes the fold of a pair of typical Fn1 modules. Gelatin affinity chromatography is used to confirm that both Fn1 and Fn2 modules contribute to gelatin binding, possibly in two clusters (1Fn2-2Fn2 and 8Fn1-9Fn1). The 7Fn1 module may perform a structural role in linking together these two interaction sites, in the same way as suggested for 6Fn1, which is thought to act in a structural manner to enhance the binding of 1Fn2-2Fn2 to gelatin. Three carbohydrate moieties are found on this domain, one on 2Fn2 and two on 8Fn1. Here, by means of expressing different protein length fragments, and by site directed mutagenesis, the role of each sugar chain is investigated independently. The sugar chain on 2Fn2 does not appear to promote binding to collagen, nor does the first sugar chain on 8Fn1 (N-linked to N497), implying another role for these sugars such as protection from proteolysis. However, the presence of at least a single GlcNAc sugar residue on the second sugar chain site on 8Fn1 (N- linked to N511) is essential for full affinity binding to collagen. Direct binding of the 8Fn1-9Fn1 module pair to collagen is assessed with a short collagen peptide and the binding is monitored by NMR. The peptide appears to bind, predominantly to the final strand of 8Fn1, the first β- strand of 9Fn1, and the linker between the two modules, with μM affinity. A model for bound peptide is proposed. The highly conserved amino acid motif Ile-Gly-Asp (IGD) is found on four of the nine N-terminal Fn1 modules of fibronectin. Tetrapeptides containing the IGD were demonstrated to promote the migration of fibroblast cells into a native collagen matrix. Two of these “bioactive” IGD motifs are found within the gelatin binding domain, one on 7Fn1 and one on 9Fn1. In this study, the motif in the 8Fn1-9Fn1 module pair is shown to be located in a tightly constrained loop within 9Fn1. By site directed mutagenesis, the IGD motifs of 7Fn1 and 9Fn1 are subjected to single amino acid substitutions, and their ability to stimulate cell migration assessed in our assay. By NMR, the fold of the IGD mutant proteins is found to be unaffected by the mutation with respect to the wild type, with the exception of small perturbations around the substitution site. While the wild type module is able to stimulate fibroblast migration, the mutant proteins show reduced or negligible bioactivity. The larger fragments show far more potency in stimulating fibroblast migration, with 8Fn1-9Fn1 (one IGD motif) 104 times more potent than the IGD peptide, and the full gelatin binding domain (two IGD motifs) 106 times more potent than the 8Fn1-9Fn1. Potential mechanisms for this enormous enhancement of the IGD potency in different contexts are discussed.

Les furocoumarines sont des phytoalexines offrant un potentiel thérapeutique important. Les travaux présentés ici portent sur les cytochromes P450 participant à leur voie de biosynthèse. Une étude «structure-fonction» de la cinnamate-4-hydroxylase (C4H) a été réalisée pour identifier les déterminants de la faible sensibilité de la C4H de Ruta graveolens (CYP73A32) au psoralène. Deux régions protéiques semblent impliquées dans l’inactivation différentielle entre CYP73A32 et CYP73A1. L’une, entre les résidus 31 et 58, est responsable de l’affinité pour le psoralène. L’autre, entre les résidus 229 et 379, contrôle la vitesse d’inactivation. La caractérisation de nouveaux P450 de la biosynthèse des furocoumarines a été entreprise. D’une part, plusieurs ADNc partiels ont été clonés chez Ruta graveolens. D’autre part, CYP71AJ1 isolé chez Ammi majus, a été caractérisé comme étant une psoralène synthase. La spécificité de CYP71AJ1 pour la marmésine a été approchée par l’étude d’un modèle 3D. Mots clés : cytochrome P450, psoralène synthase, cinnamate-4-Hydroxylase, C4H, métabolite secondaire, Ruta graveolens, Ammi majus, inactivation autocatalytique, furocoumarines, psoralène, (+)-marmésine, Modélisation 3D, (+)-columbianetine / Furocoumarins are phytoalexins known as efficient therapeutic agents. The work reported here focuses on cytochromes P450 involved in their biosynthesis pathway. A “structure-function” study was realized to understand how C4H from Ruta graveolens (CYP73A32) can resist to psoralen mechanism-based inactivation. Two parts of the protein seems involved in the differential susceptibility of CYP73A32 and CYP73A1 to psoralen. The first, between amino acids 31 and 58, defines differential affinity to psoralen. The second between residues 229 and 379 controls inactivation kinetic. The second part of this work was devoted to cloning and identification of new P450 involved in furocoumarin biosynthesis. On the one hand, several partial cDNA were cloned from Ruta graveolens. On the other hand, CYP71AJ1, cloned from Ammi majus, was identified as a psoralen synthase. The specificity of CYP71AJ1 for marmesin was approached by the study of a 3D model.

In der vorliegenden Arbeit wurde der lichtinduzierte Reaktionszyklus des bakteriellen Phytochroms Agp2 aus Agrobacterium tumefaciens mit FTIR‑ und UV‑Vis‑Spektroskopie untersucht. Der Photorezeptor besteht aus einem photosensorischen Modul und einer signalgebenden Histidin-Kinase-Domäne. Das photosensorische Modul bindet das Tetrapyrrol Biliverdin als Chromophor. Der Grundzustand von Agp2 (Pfr, 750 nm) ist gegenüber dem lichtaktivierten Zustand (Pr, 700 nm) rotverschoben, weshalb Agp2 den Bathyphytochromen zugeordnet wird. Die Untersuchungen erfolgten unter Verwendung von Isotopenmarkierung, H/D-Austauschexperimenten und ortsspezifischer Mutagenese. Daraus ließen sich folgende molekulare Änderungen charakterisieren, welche im Reaktionszyklus von Agp2 erfolgen: Die lichtinduzierte Isomerisierung des Chromophors führt zu einem Übergang vom Pfr- in den Pr-Zustand, wobei zwei Intermediate, Lumi‑F und Meta‑F, durchlaufen werden. Neben der Konformationsänderung des Chromophor‑D‑Rings ist auch die C‑Ring-Propionsäureseitenkette an der Photoreaktion beteiligt. Die C-Ring-Propionsäureseitenkette ist im Pfr-Zustand protoniert und wird im Übergang von Meta-F zu Pr deprotoniert. Der Pr-Zustand weist eine pH-Abhängigkeit auf, welche auf die pH-abhängige Ladung des Histidins 278 der Chromophortasche zurückzuführen ist. Je nach Ladung des Histidins 278 wird die Keto‑ bzw. Enolform der C(19)=O‑Gruppe des D‑Rings stabilisiert. Die Keto/Enol-Tautomerie ist auf eine innerhalb des Chromophors erfolgende Protontranslokation zurückzuführen und moduliert die Relaxation in den Pfr-Zustand. Änderungen der Amid-I-Absorption im Pfr-Pr-Übergang werden der Umstrukturierung der Tongue-Region des photosensorischen Moduls von einer Alpha-helikalen zu einer Beta‑Faltblatt-Struktur zugeordnet. Diese Strukturänderung wird als möglicher Weg der proteininternen Signaltransduktion zwischen photosensorischem und signalgebendem Modul vorgeschlagen. / In this thesis the light-induced reaction cycle of the bacterial phytochrome Agp2 from Agrobacterium tumefaciens was investigated using FTIR and UV‑vis spectroscopy. The photoreceptor comprises a photosensitive module and a signalling histidine kinase domain. The photosensitive module binds the biliverdin tetrapyrrol as chromophore. The Agp2 ground state (Pfr, 750 nm) is red-shifted in comparison with its light-activated state (Pr, 700 nm). Therefore, Agp2 is assigned to the group of bathy phytochromes. The investigations were conducted using isotopically labelled protein, labelled chromophore as well as hydrogen‑deuterium (H‑D) exchange and site-directed mutagenesis. Based on these the following molecular changes could be characterized that occur in the reaction cycle of Agp2: The light-induced isomerization of the chromophore leads to a transition from the Pfr to the Pr state, involving two intermediates, Lumi-F and Meta-F. Besides conformational changes of the chromophore D-ring, the C-ring propionic side chain is involved in the photoreaction as well. The C-ring propionic side chain is protonated in the Pfr state and gets deprotonated in the Meta-F to Pr transition. The Pr state exhibits pH‑dependent alterations which can be explained by pH dependent polarity changes of histidine 278 in the chromophore pocket. Depending on the charge of histidine, the D‑ring C(19)=O group is stabilized either in keto or enol form. The keto/enol tautomerism involves a proton translocation within the chromophore and modulates the relaxation to the Pfr state. The changes in the amide I region in the Pfr-Pr transition are associated with an alpha‑helix to beta‑sheet secondary structure change of the PHY domain tongue‑region. This structural change is proposed as the potential path of signal transduction between the photosensitive and the signalling module.

Mapeamento dos subsítios de α-amilase de Xanthomonas axonopodis pv. Citri envolvidos na interação com o substrato A família das enzimas α-amilases é um modelo experimental interessante para o estudo das interações entre os aminoácidos e seus ligantes, já que estas enzimas apresentam especificidade variável, são frequentemente alvos de estudos por mutagênese e há estruturas cristalinas disponíveis para alguns membros da família. A proposta deste trabalho foi o mapear subsítios da α-amilase de Xanthomonas axonopodis pv. citri (AXA) envolvidos na interação com substratos, através de comparações estruturais, mutagêneses sítio-dirigidas, análises de parâmetros cinéticos sobre amido e do padrão de clivagem sobre p-nitrofenil malto-oligossacarideos (PNPG7, PNPG5, PNPG4). Foi criado um modelo estrutural para AXA a partir da estrutura tridimensional da α-amilase de Alteromonas haloplanctis (Aghajari et al., 1998). O modelo de AXA foi sobreposto na estrutura da α-amilase pancreática de porco (Qian et al., 1994) e 11 resíduos foram selecionados e mutados para alanina. As α-amilases recombinantes mutantes e selvagem foram secretadas pela levedura Pichia pastoris GS115, apresentando uma massa molecular aparente de 45 kDa. Todos os mutantes analisados reduziram em maior ou menor grau a atividade catalítica da enzima sobre amido e p-nitrofenil maltooligossacarideos. Mutações dos resíduos H88, F136, D196, E223, D295 e N299, deletaram a atividade enzimática, indicando que suas cadeias laterais são essenciais para o desempenho catalítico da enzima. As análises cinéticas e estruturais sugerem fortemente que D196, E223 e D295 são os resíduos catalíticos. Substituições das cadeias laterais de C157, H200, G227, T230 e H294 reduziram a eficiência catalítica (kcat/Km) da α-amilase sobre o substrato amido para, respectivamente, 28%, 41%, 84%, 81% e 51%. As mutações em G227 e T230 foram menos importantes para a atividade da enzima e afinidade pelo amido, entretanto, estes resíduos mostraram-se importantes para a estabilização de complexos com substratos curtos (pNPG4). Os resultados indicam que o sítio ativo de AXA é formado por, no mínimo, seis subsítios. As interações dos anéis de glicose com os subsítios +2 e -2 são favorecidas em relação às interações nos subsítios -3 e +3, respectivamente, e a interação do anel de glicose no subsítio -3 é favorecida em relação à interação no subsítio +3. A enzima selvagem diva preferencialmente a terceira ligação glicosídica de p-nitrofenil maltooligossacarideos. Como produtos de hidrólise a enzima libera maltopentaose, maltotetraose, maltotriose, maltose e glicose. / The α-amylase family is an interesting group for structure/function relationship investigation, as this family exhibits a variable deavage patterm, several crystal structures are available, and its members were studied by mutagenesis. The aim of this study was the mapping of Xanthomonas axonopodis pv. Citri α-amylase (AXA) subsites involved in substrate binding, using structural comparison, site-directed mutagenesis and lcinetics analyses. A structural model for AXA was created from the three-dimensional structure of the α-amylase from Alteromonas haloplanctis (Aghajari et al., 1998). This model was superimposed on the structure ofthe pig pancreatic α-amylase, PPA (Qian et. al., 1994), and 11 residues were selected and changed to alanine. Wild type and mutant AXA were secreted by Pichia pastoris strain GS115 cells and showed apparent molecular mass of 45 kDa. All mutants have reduced α-amylase activity on starch and 4-nitrophenyl maltooligosaccharides (pNPG7, PNPG5 and PNPG4) at different levels. Mutation of residues H88, F136, D196, E223, D295 and N299 indicate their essential role by complete loss of activity. Kinetic and structural analyses strongly suggested that D196, E223 and D295 are the catalytic residues. The substitution of the side chain of C157, H200, G227, T230 and H294 reduced the catalytic efficiency (kcat/Km) of α-amylase on starch to respectively 28%, 41%, 84%, 81% and 51%. Although G227 and T230 were not much important for activity and binding on starch, these residues were important for stabilization of complexes with short substrates (PNPG4). The results indicate that AXA\'s active site is composed of at least six sugar binding subsites. The binding of the glucoses at subsites +2 and -2 are favored against binding at subsites -3 and +3, respectively. The binding of glucose at subsite -3 is favored against binding at subsite +3. The wild type enzyme primarily hydrolyzes the third glucosidic bond in PNPG7, PNPG5 and PNPG4 and the products of hydrolysis were maltopentaose, maltotetraose, maltotriose, maltose and glucose.

Glutathione transferase (GST) A2-2 is the human enzyme most efficient in catalyzing azathioprine activation. Structure-function relationships were sought explaining the higher catalytic efficiency compared to other alpha class GSTs. By screening a DNA shuffling library, five recombined segments were identified that were conserved among the most active mutants. Mutational analysis confirmed the importance of these short segments as their insertion into low-active GSTs introduced higher azathioprine activity. Besides, H-site mutagenesis led to decreased azathioprine activity when the targeted positions belonged to these conserved segments and mainly enhanced activity when other positions were targeted. Hydrophobic residues were preferred in positions 208 and 213. The prodrug azathioprine is today primarily used for maintaining remission in inflammatory bowel disease. Therapy leads to adverse effects for 30 % of the patients and genotyping of the metabolic genes involved can explain some of these incidences. Five genotypes of human A2-2 were characterized and variant A2*E had 3–4-fold higher catalytic efficiency with azathioprine, due to a proline mutated close to the H-site. Faster activation might lead to different metabolite distributions and possibly more adverse effects. Genotyping of GSTs is recommended for further studies. Molecular docking of azathioprine into a modeled structure of A2*E suggested three positions for mutagenesis. The most active mutants had small or polar residues in the mutated positions. Mutant L107G/L108D/F222H displayed a 70-fold improved catalytic efficiency with azathioprine. Determination of its structure by X-ray crystallography showed a widened H-site, suggesting that the transition state could be accommodated in a mode better suited for catalysis. The mutational analysis increased our understanding of the azathioprine activation in alpha class GSTs and highlighted A2*E as one factor possibly behind the adverse drug-effects. A successfully redesigned GST, with 200-fold enhanced catalytic efficiency towards azathioprine compared to the starting point A2*C, might find use in targeted enzyme-prodrug therapies.

Human α-N-Acetylglucosaminidase (Naglu) is a lysosomal acid hydrolase implicated in tthe rare metabolic storage disorder known as mucopolysaccharidosis type IIIB (MPS IIIB; also Sanfilippo syndrome B). Absence of this enzyme results in cytotoxic accumulation of heparan sulphate in the central nervous system, causing mental retardation and a shortened lifespan. Enzyme replacement therapy is not currently effective to treat neurological symptoms due to the inability of exogenous Naglu to access the brain. This laboratory uses a Spodoptera frugiperda (Sf9) insect cell system to express Naglu fused to a synthetic protein transduction domain with the intent to facilitate delivery of Naglu across the blood-brain barrier. The project described herein may be broken down into three main sections. Firstly, the impact of two cryptic splice sites on Naglu expression levels was analyzed in both transiently expressing Sf9 cultures and stably selected cell lines. Secondly, the effectiveness of the native Naglu secretion-signaling peptide in the Sf9 system was examined. Finally, purification of a Naglu fusion protein from suspension culture medium was performed using hydrophobic interaction chromatographic techniques. The ultimate goal of this research is to develop an efficient system for economical, large-scale production of a human recombinant Naglu fusion protein that has the potential to be successfully used for enzyme replacement therapy to treat MPS IIIB. / Graduate

Mapeamento dos subsítios de α-amilase de Xanthomonas axonopodis pv. Citri envolvidos na interação com o substrato A família das enzimas α-amilases é um modelo experimental interessante para o estudo das interações entre os aminoácidos e seus ligantes, já que estas enzimas apresentam especificidade variável, são frequentemente alvos de estudos por mutagênese e há estruturas cristalinas disponíveis para alguns membros da família. A proposta deste trabalho foi o mapear subsítios da α-amilase de Xanthomonas axonopodis pv. citri (AXA) envolvidos na interação com substratos, através de comparações estruturais, mutagêneses sítio-dirigidas, análises de parâmetros cinéticos sobre amido e do padrão de clivagem sobre p-nitrofenil malto-oligossacarideos (PNPG7, PNPG5, PNPG4). Foi criado um modelo estrutural para AXA a partir da estrutura tridimensional da α-amilase de Alteromonas haloplanctis (Aghajari et al., 1998). O modelo de AXA foi sobreposto na estrutura da α-amilase pancreática de porco (Qian et al., 1994) e 11 resíduos foram selecionados e mutados para alanina. As α-amilases recombinantes mutantes e selvagem foram secretadas pela levedura Pichia pastoris GS115, apresentando uma massa molecular aparente de 45 kDa. Todos os mutantes analisados reduziram em maior ou menor grau a atividade catalítica da enzima sobre amido e p-nitrofenil maltooligossacarideos. Mutações dos resíduos H88, F136, D196, E223, D295 e N299, deletaram a atividade enzimática, indicando que suas cadeias laterais são essenciais para o desempenho catalítico da enzima. As análises cinéticas e estruturais sugerem fortemente que D196, E223 e D295 são os resíduos catalíticos. Substituições das cadeias laterais de C157, H200, G227, T230 e H294 reduziram a eficiência catalítica (kcat/Km) da α-amilase sobre o substrato amido para, respectivamente, 28%, 41%, 84%, 81% e 51%. As mutações em G227 e T230 foram menos importantes para a atividade da enzima e afinidade pelo amido, entretanto, estes resíduos mostraram-se importantes para a estabilização de complexos com substratos curtos (pNPG4). Os resultados indicam que o sítio ativo de AXA é formado por, no mínimo, seis subsítios. As interações dos anéis de glicose com os subsítios +2 e -2 são favorecidas em relação às interações nos subsítios -3 e +3, respectivamente, e a interação do anel de glicose no subsítio -3 é favorecida em relação à interação no subsítio +3. A enzima selvagem diva preferencialmente a terceira ligação glicosídica de p-nitrofenil maltooligossacarideos. Como produtos de hidrólise a enzima libera maltopentaose, maltotetraose, maltotriose, maltose e glicose. / The α-amylase family is an interesting group for structure/function relationship investigation, as this family exhibits a variable deavage patterm, several crystal structures are available, and its members were studied by mutagenesis. The aim of this study was the mapping of Xanthomonas axonopodis pv. Citri α-amylase (AXA) subsites involved in substrate binding, using structural comparison, site-directed mutagenesis and lcinetics analyses. A structural model for AXA was created from the three-dimensional structure of the α-amylase from Alteromonas haloplanctis (Aghajari et al., 1998). This model was superimposed on the structure ofthe pig pancreatic α-amylase, PPA (Qian et. al., 1994), and 11 residues were selected and changed to alanine. Wild type and mutant AXA were secreted by Pichia pastoris strain GS115 cells and showed apparent molecular mass of 45 kDa. All mutants have reduced α-amylase activity on starch and 4-nitrophenyl maltooligosaccharides (pNPG7, PNPG5 and PNPG4) at different levels. Mutation of residues H88, F136, D196, E223, D295 and N299 indicate their essential role by complete loss of activity. Kinetic and structural analyses strongly suggested that D196, E223 and D295 are the catalytic residues. The substitution of the side chain of C157, H200, G227, T230 and H294 reduced the catalytic efficiency (kcat/Km) of α-amylase on starch to respectively 28%, 41%, 84%, 81% and 51%. Although G227 and T230 were not much important for activity and binding on starch, these residues were important for stabilization of complexes with short substrates (PNPG4). The results indicate that AXA\'s active site is composed of at least six sugar binding subsites. The binding of the glucoses at subsites +2 and -2 are favored against binding at subsites -3 and +3, respectively. The binding of glucose at subsite -3 is favored against binding at subsite +3. The wild type enzyme primarily hydrolyzes the third glucosidic bond in PNPG7, PNPG5 and PNPG4 and the products of hydrolysis were maltopentaose, maltotetraose, maltotriose, maltose and glucose.

Les maladies métaboliques, comme le diabète, la dyslipidémie ou l’obésité, constituent un problème majeur de santé publique dans les pays développés. Ces maladies très répandues restent encore difficiles à traiter malgré une recherche active. Les stratégies thérapeutiques contre ces maladies incluent le développement de nouvelles molécules ciblant les récepteurs aux acides gras, étant donné leur rôle essentiel dans l’homéostasie du métabolisme. C’est dans ce contexte que s’inscrit ce travail portant sur deux récepteurs couplés aux protéines G, les récepteurs aux acides gras à courte chaîne 2 et 3 ou free fatty acid receptors 2 (FFA2) et 3 (FFA3). Nous avons tout d'abord cherché à déterminer le profil d'expression des deux récepteurs. Ensuite, nous avons établi des lignées cellulaires stable exprimant FFA2 ou FFA3 afin d’étudier la pharmacologie d’agonistes synthétiques et endogènes de ces récepteurs. Après avoir identifié les voies de signalisation engendrées par l’activation des récepteurs, nous avons démontré que les agonistes synthétiques étaient des activateurs allostériques, c’est-à-dire qu’ils se liaient aux récepteurs sur un site distinct de celui des ligands endogènes. Pour identifier les résidus d’acides aminés nécessaires à la reconnaissance des ligands, nous avons généré une gamme de mutants ponctuels de ces récepteurs par mutagénèse dirigée. En analysant l’effet des mutations dans des tests fonctionnels, nous avons pu déterminer avec précision où se liaient les ligands et ainsi pu dessiner par informatique des modèles structuraux des récepteurs qui pourront être utilisés pour le drug design de futures molécules agonistes de ces récepteurs. / Metabolic diseases, such as diabetes, dyslipidemia or obesity, are more and more weighing on public health expenses in developed countries. Despite active research, these widespread diseases remain difficult to handle. Promising new therapeutic strategies against metabolic diseases include the development of drugs targeting the free fatty acid receptors, as key players in metabolism homeostasis. In this context, the current PhD thesis focuses on the study of two G protein-coupled receptors, namely the short-chain free fatty acid receptors 2 (FFA2) and 3 (FFA3). First, we investigated the expression of the two receptors of interest in a variety of cell types. Then, in order to study the pharmacology and the binding mode of endogenous and synthetic agonists on FFA2 and FFA3, we established stable cell lines expressing each receptor. Once we identified the signaling pathways engendered in response to receptor activation, we showed that synthetic agonists were allosteric activators of the receptors, in the sense that they bind to the receptors at a distinct site from short-chain fatty acids, i.e. the endogenous agonists. To identify the aminoacid residues that were involved in ligand binding, we generated a variety of point mutated receptors by site-directed mutagenesis. By analyzing the effects of the mutations in functional tests, we determined precisely the aminoacid residues that were essential for ligand binding. From these results, we designed in silico structural models which may aid future drug design efforts for the discovery of new FFA2 and FFA3 agonists.