Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Vitamin C (ascorbate or AsA) is a secondary metabolite produced in many eukaryotes including yeasts, plants and animals. It plays essential roles as an anti-oxidant and enzyme cofactor, functions as an electron donor and -acceptor and is involved in various developmental processes. This study was initiated with the aim of increasing vitamin C production in tomato. Three genes, namely GDP-mannose pyrophosphorylase (GMPase) from Saccharomyces cerevisiae, arabinono-1,4-lactone oxidase (ALO) from Saccharomyces cerevisiae and myo-inositol oxygenase 2 (MIOX2) from Arabidopsis thaliana were ectopically expressed in the tomato cultivar Money Maker. GMPase converts D-mannose-6-P to GDP-D-mannose. This reaction forms part of the well characterized, “Smirnoff-Wheeler” pathway. ALO catalyzes the terminal step in erythroascorbate synthesis in yeast. In situ it also metabolizes the plant and animal substrates for ascorbate manufacture. Myo-inositol (MI) is converted into D-glucuronate by the activity of MIOX. D-Glucuronate is a precursor to L-guluno-1,4-lactone synthesis which is the precursor to AsA in animals and thought to be present in plants. The genes were independently introduced with the aid of Agrobacterium tumefaciens mediated transformation and expressed under the control of the CaMV 35S promoter. Plants with increased GMPase activity consistently showed increased L-ascorbate levels in leaves and fruit of between 20- and 70% compared to the wild-type. Plants transcribing the ALO gene exhibited small increases in L-ascorbate in green fruit (p < 0.1). Leaf tissue from MIOX plants displayed significant activity increases (p < 0.05), and substantial decreases in MI. In green fruit two MIOX lines had increases in activity, cell wall uronic acids and AsA levels. Marginal increases in L-ascorbate would not warrant industrial application, but follow-up research with over-expression of other enzymes of the “Smirnoff-Wheeler” pathway should be explored. / AFRIKAANSE OPSOMMING: Vitamien C (askorbiensuur of AsA) is ʼn sekondêre metaboliet wat in baie eukariote, insluitend gis, plante en diere geproduseer word. Dit speel ʼn noodsaaklike rol as ʼn anti-oksidant en ensiem kofaktor, funktioneer as ʼn elekronskenker en aanvaarder en is betrokke in verskillende ontwikkelings prosesse. Hierdie studie was geїnisieer met die doel om vitamien C produksie in tamatie te vermeerder. Drie gene, naamlik GDP-mannose pirofosforilase (GMPase) van Saccharomyces cerevisiae, arabinono-1,4-laktoon oksidase (ALO) van Saccharomyces cerevisiae en mio-inositol oksigenase 2 (MIOX2) van Arabidopsis thaliana was ektopies uitgedruk in the tamatie kultivar, Money Maker. GMPase skakel D-mannose-6-P om na GDP-D-mannose. Hierdie reaksie is deel van die goed gekenmerkde “Smirnoff Wheeler” baan. ALO kataliseer the terminale stap in eritroaskorbiensuur sintese in gis. In situ metaboliseer dit ook die plant en dier substrate om askorbiensuur te vervaardig. Mio-inositol (MI) is omgeskakel na D-glukuronsuur deur die aktiwiteit van MIOX. D-glukuronsuur is ʼn voorloper in L-guluno-1,4-laktoon sintese wat dan ʼn voorloper is van AsA in diere en word ook verdink om in plante teenwoordig te wees. Die gene was onafhanklik ingestel met die hulp van Agrobakterium tumefaciens gemedїeerde transformasie en uitgedruk onder die beheer van die CaMV 35S promotor. Plante met verhoogde GMPase aktiwiteit het in blare en vrugte konsekwente toename in L-askorbiensuur vlakke met tussen 20 – 70% gewys in vergelyking met wilde-tipe. Plante wat ALO getranskribeer het, het klein stygings in L-askorbiensuur in groen vrugte gewys (p < 0.1). Blaarweefsel van MIOX plante wat verhoogde aktiwiteit vertoon het, (p < 0.05), het ook aansienlike dalings in MI gehad. In groen vrugte van MIOX het twee lyne verhoogte aktiwiteit, selwand uronsuur en AsA vlakke gehad. Klein toename in L-askorbiensuur is nie gepas vir industriële toepassing nie, maar opvolg navorsing moet ondersoek word met die oor-uitdrukking van ander “Smirnoff-Wheeler” baan ensieme.

Le coenzyme Q est une molécule lipophile rédox rencontrée chez les eucaryotes et chez la plupart des procaryotes. La structure de Q correspond à une benzoquinone substituée par une chaîne polyisoprényle dont la longueur varie selon les organismes. Q joue le rôle de transporteur d'électrons dans les chaînes respiratoires d'où provient la plupart de l'énergie de la cellule. La biosynthèse de Q chez la bactérie Escherichia coli comporte huit étapes et implique au moins neuf protéines (UbiA-UbiH et UbiX). Trois réactions d'hydroxylation sont nécessaires pour la biosynthèse de Q8 en conditions aérobies. Alors que les protéines UbiH et UbiF présentent des homologies de séquence avec des monooxygénases à flavine connues pour catalyser des réactions d'hydroxylation, UbiB qui a été proposée comme étant la troisième hydroxylase, présente uniquement une homologie de séquence avec des kinases. Nous rapportons dans ce travail que la protéine VisC, renommée UbiI, catalyse la réaction d'hydroxylation auparavant attribuée à UbiB. Nous avons également identifié deux nouvelles protéines (YigP et YqiC, renommées respectivement UbiJ et UbiK) importantes pour le métabolisme de Q chez Escherichia coli puisque leur mutation diminue fortement le contenu en Q des souches mutantes. Ces protéines interagissent avec la plupart des protéines connues pour participer à la biosynthèse de Q ce qui implique l'existence d'un complexe de biosynthèse de Q. En utilisant des approches biochimiques et protéomiques, nous avons pu mettre en évidence un complexe impliquant plusieurs protéines Ubi et notamment UbiJ et UbiK. Ces deux protéines semblent avoir un rôle dans l'assemblage et/ou la stabilisation de ce complexe multiprotéique. Enfin, nous nous sommes intéressés à la biosynthèse de Q dans des conditions de cultures anaérobies. Nos résultats montrent l'existence « d'hydroxylases anaérobies », inconnues à ce jour, qui remplaçent les hydroxylases aérobies UbiH, UbiI et UbiF. Grâce à une approche phylogénétique, nous identifions un gène important pour la biosynthèse de Q uniquement en conditions anaérobies suggérant une réorganisation de la biosynthèse de Q entre ces deux environnements fréquemment rencontrés par E. coli. L'ensemble de nos résultats a permis d'améliorer notre connaissance de la voie de biosynthèse procaryote de Q grâce à la découverte de nouveaux gènes impliqués dans ce processus et grâce à l'identification de la fonction moléculaire de certaines protéines. / Ubiquinone (Q) is a lipophilic compound that plays an important role in electron and proton transport in the respiratory chains of Escherichia coli. Besides this important role in energy production, Q also functions as a membrane soluble antioxidant. The biosynthesis of Q8 requires eight reactions and involves at least nine proteins (UbiA-UbiH and UbiX) in Escherichia coli. Three of these reactions are hydroxylations resulting in the introduction of a hydroxyl group on carbon atoms at position 1, 5 and 6 of the aromatic ring. The C1 and C6 hydroxylation are well characterized whereas the C5 hydroxylation has been proposed to involve UbiB, a protein kinase without any sequence homology with monooxygenase. In this work, by genetic and biochemical methods we provide evidence that VisC which we renamed UbiI, displays sequence homology with monooxygenases and catalyzes the C5 hydroxylation, not UbiB. We have identified two new genes, yqiC and yigP (renammed UbiJ and UbiK) which are required only for Q8 biosynthesis in aerobic conditions. The exact role of the corresponding proteins, renamed UbiJ and UbiK, remains unknown. These proteins are able to interact with other Ubi proteins to be able to produce Q supporting the protein complex hypothesis. Our progress on the characterization of an Ubi-complex regrouping several Ubi proteins suggest that UbiJ and UbiK may fulfill functions related to the Ubi-complex stability. Mutants affected in hydroxylation steps are deficient for Q8 in aerobic conditions but recover a wild type Q8 content when grown in anaerobic conditions. This intriguing observation supports the existence of an alternative hydroxylation system independent from dioxygen which has not been characterized so far. By phylogenetic studies, we have identified a new gene in which the deletion affect the biosynthesis of Q only in anaerobic conditions suggesting a reorganization of Q biosynthesis in these two conditions. Our results has improved our knowledge of the prokaryotic Q biosynthetic pathway through the discovery of new genes involved in this process and through the identification of the molecular function of some proteins.

Nonsense-mediated mRNA decay (NMD) is an important mRNA surveillance mechanism conserved in eukaryotes. This thesis explores several interesting aspects of the NMD pathway. One important aspect of NMD which is presently the subject of intense controversy is the subcellular localization of NMD. In one set of experiments, the decay kinetics of the ade2-1 and pgk1 nonsense mRNAs (substrates for NMD) were investigated in response to activating the NMD pathway to determine if cytoplasmic nonsense mRNAs are immune to NMD in the yeast system. The results of these studies demonstrated that activation of NMD caused rapid and immediate degradation of both the ade2-1 and the early nonsense pgk1 steady state mRNA populations. The half lives of the steady state mRNA populations for both ade2-1 and pgk1 (early nonsense) were shortened from >30 minutes to approximately 7 minutes. This was not observed for pgk1mRNAs that contained a late nonsense codon demonstrating that activation of NMD specifically targeted the proper substrates in these experiments. Therefore, in yeast, nonsense mRNAs residing in the cytoplasm are susceptible to NMD. While these findings are consistent with NMD occurring in the cytoplasm, they do not completely rule out the possibility of a nuclear-associated decay mechanism. To investigate the involvement of the nucleus in NMD, the putative nuclear targeting sequence identified in Nmd2p (one of the trans-acting factors essential for NMD) was characterized. Subcellular fractionation experiments demonstrated that the majority of Nmd2p localized to the cytoplasm with a small proportion detected in the nucleus. Specific mutations in the putative nuclear localization signal (NLS) of Nmd2p were found to have adverse effects on the protein's decay function. These effects on decay function, however, could not be attributed to a failure in nuclear localization. Therefore, the residues that comprise the putative NLS of Nmd2p are important for decay function but do not appear to be required for targeting the protein to the nucleus. These results are in accordance with the findings above which implicate the cytoplasm as an important cellular compartment for NMD. This thesis then investigates the regulatory roles of the trans-acting factors involved in NMD (Upf1p, Nmd2p, and Upf3p) using a novel quantitative assay for translational suppression, based on a nonsense allele of the CAN1 gene (can1-100). Deletion of UPF1, NMD2, or UPF3 stabilized the can1-100 transcript and promoted can1-100 nonsense suppression. Changes in mRNA levels were not the basis of suppression, however, since deletion of DCP1 or XRN1 or high-copy can1-100 expression in wild-type cells caused mRNA stabilization similar to that obtained in upf/nmd cells but did not result in comparable suppression. can1-100 suppression was highest in cells harboring a deletion of UPF1, and overexpression of UPF1 in cells with individual or multiple upf/nmd mutations lowered the level of nonsense suppression without affecting the abundance of the can1-100 mRNA. These findings indicate that Nmd2p and Upf3p regulate Upf1p activity and that Upf1p plays a critical role in promoting termination fidelity that is independent of its role in regulating mRNA decay.

Cyclic peptides possess desirable characteristics as potential pharmaceutical scaffolds. The cyanobactin family of cyclic peptide natural products boast diverse structures and bioactivity. Exemplars are the patellamides, which have attracted attention due to their ability to reverse the effects of multi-drug resistance in human leukemia cells. In addition to their macrocyclic architecture patellamides contain azol(in)e heterocycles and d-amino acids. This structural complexity makes them challenging targets for chemical synthesis. Understanding their biosynthesis will enable the development of a biotechnological ‘toolkit' for the synthesis of new pharmaceutical compounds. Patellamides are ribosomally-synthesised and post-translationally modified peptides (RiPPs) and much of their biosynthesis has been elucidated, however there are still elements of their biosynthesis that are not yet fully understood. PatA and PatG contain C-terminal domains of unknown function (DUFs). The crystal structure of PatG-DUF has been solved and subsequent to biochemical and biophysical investigation PatG-DUF was found not to constitute an essential part of the biotechnological ‘toolkit' and can be excluded from in vitro enzyme-based synthesis of cyanobactin-like cyclic peptides. The cyanobactin heterocyclases are able to introduce heterocycles into a peptide backbone, seemingly irrespective of the neighbouring residues; however a molecular rational governing substrate recognition is unknown. Additionally the mechanism of heterocyclisaton is disputed. Analysis of crystal structures of LynD in complex with cofactor and substrate (solved by Dr Jesko Koehnke) enabled the active site and substrate recognition site to be located. A new mechanism for heterocyclisation has been proposed. Guided by the substrate recognition observed in complex structures a constituently active heterocyclase (AcLynD) has been engineered, which is able to process short, leaderless peptide substrates. Epimerisation in cyanobactin biosynthesis is believed to be spontaneous, but its precise timing is uncertain. NMR analysis of selectively labelled peptide substrates processed by the modifying enzymes, identified epimerisation to be spontaneous on the macrocycle, regardless of whether the neighbouring heterocycles have been oxidised. A one-pot in vitro synthesis of cyanobactins has been developed, and employed to create a number of patellamide D analogues to ascertain structural-activity relationships.

ABCB10, or ATP binding cassette sub-family B member 10, is a protein localized in the mitochondrial inner membrane. It belongs to the ABC transporter family whose members are proteins that facilitate substrate transport across various biological membranes. It has been found that ABCB10 is required for normal heme biosynthesis during erythroid differentiation and also plays a role in protection against the damage caused by reactive oxygen species (ROS) production. This protective effect exists both in the erythrocyte development and in the heart recovery after the ischemia-reperfusion injury. However, as an ABC transporter, its transported substrates are not known, neither is the mechanism by which ABCB10 plays a role in protection against ROS damage. In this dissertation an 8-azido-ATP photolabeling system is established to study the ATP binding and hydrolysis properties of ABCB10. Using this approach, it is found that the conserved amino acid residues Gly497 and Lys498 in the Walker A motif of the nucleotide binding domain of ABCB10 are required for ATP binding. On the other hand, Gly602 in the C-loop motif and Glu624 in the end of the Walker B motif are necessary for ATP hydrolysis. In addition, most ABC transporters increase ATP hydrolysis in the presence of their substrates. Therefore, the 8-azido-ATP photolabeling system can be utilized to test potential substrates of ABCB10. Substances related to the heme biosynthesis such as &#948;-aminolevulinic acid (dALA) and the mitochondrial redox state such as oxidized glutathione (GSSG) and reduced glutathione (GSH) are tested for this purpose. The 8-azido-ATP photolabeling system shows that GSSG stimulates ATP hydrolysis without affecting ATP binding, whereas GSH decreases ATP binding. Further study shows that the nucleotide binding domain of ABCB10 is glutathionylated at the cysteine residue on the position 547 (Cys547), suggesting that GSH may modulate ABCB10 activity via the glutathionylation-regulated ATP binding. This is a first insight into the molecular mechanism by which the mitochondrial redox state, through the regulation by GSH and GSSG, can modulate ABCB10 activity. / 2016-09-01T00:00:00Z

The synthesis of nanostructured materials, especially metallic nanoparticles, has accrued utmost interest over the past decade owing to their unique properties that make them applicable in different fields of science and technology. The limitation to the use of these nanoparticles is the paucity of an effective method of synthesis that will produce homogeneous size and shape nanoparticles as well as particles with limited or no toxicity to the human health and the environment. The biological method of nanoparticle synthesis is a relatively simple, cheap and environmentally friendly method than the conventional chemical method of synthesis and thus gains an upper hand. The biomineralization of nanoparticles in protein cages is one of such biological approaches used in the generation of nanoparticles. This method of synthesis apart from being a safer method in the production of nanoparticles is also able to control particle morphology. In this study, a comparative biological synthesis, characterization and biomedical effects of metallic nanoparticles of platinum, gold and silver were investigated. Metallic nanoparticles were biologically synthesized using cage-like (apoferritin), barrel-like (GroEL) and non-caged (ribonuclease) proteins. Nanoparticles generated were characterized using common techniques such as UV-visible spectroscopy, scanning and transmission electron microscopy, inductively coupled optical emission spectroscopy, Fourier transform infra-red spectroscopy and energy dispersion analysis of X-rays (EDAX). Nanoparticles synthesised biologically using apoferritin, GroEL and RNase with exhibited similar chemical and physical properties as thoses nanoparticles generated chemically. In addition, the metallic nanoparticles fabricated within the cage-like and barrel-like cavities of apoferritin and GroEL respectively, resulted in nanoparticles with relatively uniform morphology as opposed to those obtained with the non-caged ribonuclease. The enzymatic (ferroxidase) activity of apoferritin was found to be greatly enhanced with platinum (9-fold), gold (7-fold) and silver (54-fold) nanoparticles. The ATPase activity of GroEL was inhibited by silver nanoparticles (64%), was moderately activated by gold nanoparticles (47%) and considerably enhanced by platinum nanoparticles (85%). The hydrolytic activity of RNase was however, lowered by these metallic nanoparticles (90% in Ag nanoparticles) and to a higher degree with platinum (95%) and gold nanoparticles (~100%). The effect of synthesized nanoparticles on the respective enzyme activities of these proteins was also investigated and the potential neurotoxic property of these particles was also determined by an in vitro interaction with acetylcholinesterase. Protein encapsulated nanoparticles with apoferrtin and GroEL showed a decreased inhibition of acetylcholinesterase (<50%) compared with nanoparticles attached to ribonuclease (>50%). Thus, it can be concluded that the cavities of apoferitin and GroEL acted as nanobiofactories for the synthesis and confinement of the size and shape of nanoparticles. Furthermore, the interior of these proteins provided a shielding effect for these nanoparticles and thus reduced/prevented their possible neurotoxic effect and confirmed safety in their method of production and application. The findings from this study would prove beneficial in the application of these nanoparticles as a potential drug/drug delivery vehicle for the prevention, treatment/management of diseases associated with these enzymes/proteins.

Made available in DSpace on 2014-06-11T19:35:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-23Bitstream added on 2014-06-13T18:46:23Z : No. of bitstreams: 1 pivatto_m_dr_araiq.pdf: 3895401 bytes, checksum: 8f00934510e296fa101a80060a9db12a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O presente trabalho teve como objetivo o estudo das vias biossintéticas dos alcalóides piperidínicos presentes em Senna spectabilis, motivado pela potente atividade anticolinesterásica e baixa toxicidade observada no derivado (–)-3-O-acetil-espectalina (15), eleito como composto líder para o desenvolvimento de fármacos anti-Alzheimer que estão em fase de estudos pré-clínicos. Por outro lado, o interesse acadêmico no conhecimento das vias metabólicas, pode levar a estudos futuros de engenharia genética para potencializar a produção desses metabólitos uma vez que a síntese é extremamente complexa em função da presença de três centros estereogênicos. Tendo isso em vista, foram selecionadas seis espécies de Senna e Cassia para avaliar a presença dos alcalóides e selecionar aquela que os produz em maior quantidade. Foram estudadas as flores de S. spectabilis, S. multijuga, S. macranthera, S. velutina, C. fistula, C. leptophylla, sendo que só foram detectados alcalóides piperidínicos e piridínicos em S. spectabilis e S. multijuga, respectivamente, utilizando a espectrometria de massas tandem. Embora sejam de classes diferentes, esses metabólitos têm padrão de substituição similar, porém, apresentaram atividade anticolinesterásica diferenciada. De S. spectabilis foram isolados os alcalóides piperidínicos: (–)-cassina (1), (–)-espectalina (9), (–)-3-O-acetil-espectalina (15), (–)-3-O-acetil-cassina (16) e identificados 7-hidroxi-carnavalina (71), 7-hidroxi-cassina (18) e/ou espicigerina (42) utilizando a EM. De S. multijuga foram isolados os alcalóides piridínicos: 7'-multijuguinona (67) e 12'-hidroxi-7'-multijuguinona (69) e identificados 7'-multijuguinol (68) e 12'-hidroxi-7'- multijuguinol (70). Para os estudos biossintéticos dos alcalóides piperidínicos foi inicialmente proposta a biogênese onde lisina e acetato foram eleitos potenciais... / The following work encompass as main goal the study of biosynthetic pathways to produce piperidine alkaloids using Senna spectabilis as natural matrix. Such research was instigated due to the high acetylcholinesterase activity and low toxicity showed by the derivative (–)-3-O-acetyl-spectaline (15), selected as a lead compound against Alzheimer’s disease and currently under pre-clinical trials. Still yet, the academic interest on researching metabolic pathways that may lead to further genetic engineering studies to enhance the production of these metabolites is of extremely importance, due to the inability of producing any commercially viable synthetic strategy for their stereogenic centers. We selected six Senna and Cassia species to evaluate the presence of these metabolites aiming to select which matrix will produce them the most. We studied flowers from S. spectabilis, S. multijuga, S. macranthera, S. velutina, C. fistula and C. leptophylla. From those, we were able to detect piperidine and pyridine alkaloids only in S. spectabilis and S. multijuga, respectively, using tandem mass spectrometry. Regardless of the different structural nature towards 15, those metabolites have similar substitution patterns and showed differential acetylcholinesterase activity. From S. spectabilis were isolated the piperidine alkaloids: (–)-cassine (1), (–)-spectaline (9), (–)-3-O-acetyl-spectaline (15), (–)-3-O-acetyl-cassine (16), and identified 7-hidroxy- carnavaline (71), 7-hidroxy-cassine (18) and/or spicigerine (42) by tandem mass spectrometry and from S. multijuga were isolated the pyridine alkaloids: 7'-multijuguinone (67), 12'-hydroxy-7'-multijuguinone (69) and, identified by MS: 7'-multijuguinol (68) and 12'-hydroxy-7'-multijuguinol (70). We initially proposed the incorporation of lysine and acetate as main precursors of the piperidine alkaloids biosynthetic pathway and thus... (Complete abstract click electronic access below)

Resumo: O presente trabalho teve como objetivo o estudo das vias biossintéticas dos alcalóides piperidínicos presentes em Senna spectabilis, motivado pela potente atividade anticolinesterásica e baixa toxicidade observada no derivado (-)-3-O-acetil-espectalina (15), eleito como composto líder para o desenvolvimento de fármacos anti-Alzheimer que estão em fase de estudos pré-clínicos. Por outro lado, o interesse acadêmico no conhecimento das vias metabólicas, pode levar a estudos futuros de engenharia genética para potencializar a produção desses metabólitos uma vez que a síntese é extremamente complexa em função da presença de três centros estereogênicos. Tendo isso em vista, foram selecionadas seis espécies de Senna e Cassia para avaliar a presença dos alcalóides e selecionar aquela que os produz em maior quantidade. Foram estudadas as flores de S. spectabilis, S. multijuga, S. macranthera, S. velutina, C. fistula, C. leptophylla, sendo que só foram detectados alcalóides piperidínicos e piridínicos em S. spectabilis e S. multijuga, respectivamente, utilizando a espectrometria de massas tandem. Embora sejam de classes diferentes, esses metabólitos têm padrão de substituição similar, porém, apresentaram atividade anticolinesterásica diferenciada. De S. spectabilis foram isolados os alcalóides piperidínicos: (-)-cassina (1), (-)-espectalina (9), (-)-3-O-acetil-espectalina (15), (-)-3-O-acetil-cassina (16) e identificados 7-hidroxi-carnavalina (71), 7-hidroxi-cassina (18) e/ou espicigerina (42) utilizando a EM. De S. multijuga foram isolados os alcalóides piridínicos: 7'-multijuguinona (67) e 12'-hidroxi-7'-multijuguinona (69) e identificados 7'-multijuguinol (68) e 12'-hidroxi-7'- multijuguinol (70). Para os estudos biossintéticos dos alcalóides piperidínicos foi inicialmente proposta a biogênese onde lisina e acetato foram eleitos potenciais... (resumo completo, clicar acesso eletrônico abaixo) / Abstract: The following work encompass as main goal the study of biosynthetic pathways to produce piperidine alkaloids using Senna spectabilis as natural matrix. Such research was instigated due to the high acetylcholinesterase activity and low toxicity showed by the derivative (-)-3-O-acetyl-spectaline (15), selected as a lead compound against Alzheimer's disease and currently under pre-clinical trials. Still yet, the academic interest on researching metabolic pathways that may lead to further genetic engineering studies to enhance the production of these metabolites is of extremely importance, due to the inability of producing any commercially viable synthetic strategy for their stereogenic centers. We selected six Senna and Cassia species to evaluate the presence of these metabolites aiming to select which matrix will produce them the most. We studied flowers from S. spectabilis, S. multijuga, S. macranthera, S. velutina, C. fistula and C. leptophylla. From those, we were able to detect piperidine and pyridine alkaloids only in S. spectabilis and S. multijuga, respectively, using tandem mass spectrometry. Regardless of the different structural nature towards 15, those metabolites have similar substitution patterns and showed differential acetylcholinesterase activity. From S. spectabilis were isolated the piperidine alkaloids: (-)-cassine (1), (-)-spectaline (9), (-)-3-O-acetyl-spectaline (15), (-)-3-O-acetyl-cassine (16), and identified 7-hidroxy- carnavaline (71), 7-hidroxy-cassine (18) and/or spicigerine (42) by tandem mass spectrometry and from S. multijuga were isolated the pyridine alkaloids: 7'-multijuguinone (67), 12'-hydroxy-7'-multijuguinone (69) and, identified by MS: 7'-multijuguinol (68) and 12'-hydroxy-7'-multijuguinol (70). We initially proposed the incorporation of lysine and acetate as main precursors of the piperidine alkaloids biosynthetic pathway and thus... (Complete abstract click electronic access below) / Orientador: Vanderlan da Silva Bolzani / Coorientador: Maysa Furlan / Banca: Ian Castro-Gamboa / Banca: Frederico Guaré Cruz / Banca: Márcia Nasser Lopes / Banca: Maria Claudia Marx Young / Doutor

O estudo fitoquímico das folhas de Ocotea catharinensis (Lauraceae) resultou no isolamento da neolignana tetraidrofurânica veraguensina (1) e do flavonóide glicosilado afzelina (10), ainda não descritas para a espécie, além de quatro neolignanas hexaidrobenzofurânicas (2-5) anteriormente descritas para mesma. Nos embriões somáticos in vitro constatou-se o acúmulo de duas neolignanas hexaidrobenzofurânicas (2, 3) e duas biciclo[3.2.1]octânicas (6, 7), dois sesquiterpenos (8, 9) e um fenilpropanóide (11). A neolignana biciclo[3.2.1]octânica (7S,8R,1\'R,2\'R,3\'R)-2\'-acetoxi-3,4-metilenodioxi-3\',5\'-dimetoxi-4\'-oxo-&#916;1,3,5,5\',8\'-8.1\',7.3\'-neolignana (7), o sesquiterpeno (-)-eudesm-11-em-4&#945;-ol (9) e o fenilpropanóide 6-metóxieugenol (11) não haviam sido isolados anteriormente desta espécie. O perfil dos metabólitos secundários incluiram análises do óleo essencial das folhas cuja análise indicou a predominância de mono- e sesquiterpenos e ausência de fenilpropanóides. Além disso, foram avaliadas atividades antifúngica e antioxidante nos extratos e substâncias isoladas. A fração de CHCl3 do extrato etanólico mostrou atividade antifúngica contra Cladosporium cladosporioide ou C. sphaerospermum. As frações de CHCl3 e de AcOEt do extrato etanólico, as neolignanas hexaidrobenzofurânica (5), biciclooctânica (6), o flavonóide glicosilado (10) e o fenilpropanóide (11) apresentaram atividade antioxidante através do método de DPPH (1,1-difenil-2-picril-hidrazila). A avaliação de hipóteses biossintéticas envolvidas na formação de neolignanas em O. catharinensis fizeram uso de culturas embriogênicas como modelo experimental. Foram realizados diversos estudos de bioconversões de precursores como o isoeugenol e 5-metoxieugenol utilizando-se suspensões celulares e frações enzimáticas porém as conversões in vivo utilizando-se precursores marcados e os embriões foram melhor sucedidas. Entre os precursores radioativos, a L-[U-14C]-fenilalanina foi incorporada às neolignanas hexaidrobenzofurânicas (2, 0,30% e 3, 0,19%) e biciclooctânica (6, 0,12%). No caso do ácido [8-14C]-ferúlico, esse foi incorporado à neolignana hexaidrobenzofurânica (2, 0,17%). Por outro lado, o uso de L-[1-13C]-fenilalanina e análise por espectrometria de massas-massas confirmou o enriquecimento de 13C nas neolignanas hexaidrobenzofurânicas (2 e 3). A análise por RMN de 13C indicou o enriquecimento de 4,3 e 5,0% nos carbonos C9 e C9\', respectivamente. Desta maneira, através dos dados de incorporação desses precursores, desvenda-se parte da via biossintética de neolignanas em O. catharinensis. / The phytochemical study of Ocotea catharinensis (Lauraceae) leaves resulted in the isolation of tetrahydrofuran neolignan veraguensin (1) and glycosylated flavonoid afzelin (10), not yet described for this species, besides four hexahydrobenzofuran neolignans (2-5), which had been previously described. The in vitro somatic embryos showed the accumulation of two hexahydrobenzofuran (2, 3) and two bicyclo[3,2,1]octane (6, 7) neolignans, two sesquiterpenes (8, 9) and one phenylpropanoid (11). Among these compounds (7S,8R,1\'R,2\'R,3\'R)-2\'-acetoxy-3,4-methylenedioxy-3\',5\'-dimethoxy-4\'-oxo-&#916;1,3,5,5\',8\'-8.1\',7.3\'-neolignan (7), (-)-eudesm-11-en-4&#945;-ol (9) and 6-methoxy-eugenol (11) have not been previously described for this species. The profile of secondary metabolite included the analysis of essential oil of leaves which indicated the predominance of mono- and sesquiterpene and no phenylpropanoids. In addition, antifungal and antioxidative activities were performed with extracts and isolated compounds. The CHCl3 fraction of ethanol extract exhibited antifungal activities against Cladosporium cladosporioide or C. sphaerospermum. The CHCl3 and EtOAc fractions of ethanol extract, the hexahydrobenzofuran (5) and a bicyclo[3.2.1]octane (6) neolignans, afzelin (10) and 6-methoxy-eugenol (11) displayed antioxidant activities using DPPH (1,1-diphenyl-2-picryl-hydrazyl). The evaluations of biosynthetic hypothesis for neolignan formation in O. catharinensis were based on the use of embriogenic culture as an experimental model. Several assessment for conversion of putative precursors such as eugenol and 5-methoxyeugenol were evaluated using suspension cells and cell free preparations but none of them were as effective as in vivo conversion using labeled precursors directly in embryos. Among the radioactive precursors L-[U-14C]-phenylalanine was incorporated to hexahydrobenzofuran (2, 0.30%; 3, 0.19%) and bicyclo[3.2.1]octane (6, 0.17%) neolignans while [8-14C]-ferulic acid was incorporated only to the hexahydrobenzofuran neolignan (2, 0,17%). The tandem mass spectrometry analysis confirmed the incorporation of 13C atoms in the neolignans (2, 3) indicating the incorporation of one or two molecules of L-[1-13C]-phenylalanine. The analysis of 13C NMR data revealed the enrichment of 4.3 and 5.0% at the positions C9 and C9\', respectively. In summary, the labeling experiments have contributed to unravel the biosynthesis of neolignans in O. catharinensis.

O estudo fitoquímico de Peperomia oreophila revelou a presençca de duas lignanas furofurânicas (7R, 8R, 7R, 8R)-3,4,5-trimetóxi-3,4-metilenodioxi-5-metóxi- 8.8,7.O.9,7.O.9-lignana (1), (7R, 8R, 7R, 8R)-3,4,5-trimetóxi-3,4,5-trimetóxi- 8.8,7.O.9,7.O.9-lignana (2); as duas amidas (2E)-N-isobutil-3-(5-metóxi-7,8- benzodioxol-1-il)acrilamida (3), (2E)-N-isobutil-3-(3,4,5-trimetóxifenil)acrilamida (4), três derivados de acido cinâmico (2E)-3-(3,4,5-trimetóxifenil)acrilato de metila (5), (2Z)-3- (3,4,5-trimetóxifenil)acrilato de metila (6), (2E)-3-(5-metóxi-7,8-benzodioxol-1-il)acrilato de metila (7); os dois policetídeos fenólicos [(2E)-3,7-dimetilocta-2,6-dien-1-il]-5- metil-2-(3-metilbut-2-en-1-il)benzeno-1,3-diol (8) (inédita) e [(2E)-3,7-dimetilocta- 2,6-dien-1-il]-2,2,7-trimetil-2H-cromen-5-ol (9); de P. arifolia: o policetídeo fenólico [(2E)-3,7-dimetilocta-2,6-dien-1-il]-5-metil-2-(3-metilbut-2-en-1-il) benzeno-1,3-diol, isolada também de P. oreophila (10) (inédita); de P. urocarpa: o policetídeo fenólico 5- metil-2-[(2E,6E)-3,7,11-trimetildodeca-2,6,10-trien-1-il] benzeno-1,3-diol (11) e o ácido 2,4-dihidróxi-6-metil-3-[(2E,6E)-3,7,11-trimetildodeca-2,6,10-trien-1-il] benzóico, (12); de P. nitida: o fenilpropanoide apiol (1-alil-3,6-dimetóxi-10,11- benzodioxol) (13), os cromenos 7-hidróxi-2,2,5-trimetil-2H-cromeno-carboxilato de metila (14) e o 7-metóxi-2,2,5-dimetil-2H-cromeno-6-carboxilato de metila (15). O policetídeo 2-hidróxi-4,6-dimetóxiacetofenona, principal metabólito das folhas de P. glabella, teve sua biossíntese investigada utilizando-se como precursores o acetil-CoA e o malonil-CoA. Foram realizados estudos de otimização da atividade de policetídeo sintase (PKS) em função do pH, tempo de reação, temperatura e saturação de substratos, além de estudos da variação circadiana. Estudos de genes de PKS resultaram em amplificações cujo seqüenciamento poderá determinar a identidade dessas regiões e homologia entre as seqüências dessas Peperomias e a região KS do gene AviM de Streptomyces viridochromogenes que expressa o ácido orselínico / The phytochemical investigation carried out on Peperomia oreophila revealed the accumulation of two furofuran lignans (7R,8R,7R,8R)-3,4,5-trimethoxy-3,4- methylenedioxy-8.8, 7.O.9, 9.O.7-lignan (1), (7, 8R, 7R, 8)-3,4,5-trimethoxy-3,4,5- trimethoxy-8.8-7.O.9, 9.O.7-lignan (2); two amides (2´E)-N-isobutyl-3´-(5-methoxy-7,8- benzodioxol-1-yl) acrylamide (3), (2E)-N-isobutyl-3-(3,4,5-trimethoxyphenyl)acrylamide (4), three derivate cinâmic acid methyl (2E)-3-(3,4,5-trimethoxyphenyl)acrylate (5), methyl (2Z)-3-(3,4,5-trimethoxyphenyl)acrylate (6), methyl (2E)-3-(5-methoxy-7,8- benzodioxol-1-yl)acrylate (7); two phenolic polyketides [(2E)-3,7-dimethylocta-2,6- dien-1-yl]-5-methyl-2-(3-methylbut-2-en-1-yl)benzene-1,3-diol (8) (novel), [(2´E)-3´,7´- dimethylocta-2´,6´-dien-1-yl]-2,2,7-trimethyl-2H-chromen-5-ol (9); P. arifolia, two phenolic polyketide [(2E)-3,7-dimethylocta-2,6-dien-1-yl]-5-methyl-2-(3-methylbut-2- en-1-yl)benzene-1,3-diol, also isolated from P. oreophila (10) (novel); P. urocarpa, the two phenolic polyketides 5-methyl-2-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1- yl]benzene-1,3-diol (11) and 2,4-dihydroxy-6-methyl-3-[(2E,6E)-3,7,11-trimethyldodeca- 2,6,10-trien-1-yl]benzoic acid (12); P. nitida, the phenylpropanoid apiole 1-allyl-3,6- dimethoxy-10,11-benzodioxole (13); the chromenes methyl 7-hydroxy-2,2,5-trimethyl- 2H-chromene-6-carboxylate (14) and methyl 7-methoxy-2,2,5-trimethyl-2H-chromene-6- carboxylate (15). The polyketide 2-hydroxy-4,6-dimethoxyacetophenone, the major compound in P. glabella leaves, had its biosynthetic origin investigated using acetyl-CoA and malonyl-CoA as precursors. The enzymatic activity of polyketide synthase was optimized to pH, incubation time, temperatures and substrate saturation, in addition to the analysis of circadian variation activity. The amplifications of putative PKS genes was based on primers from AviM gene of Streptomyces viridochromogenes that express for orsellinic acid. The sequencing will enable the identification of such regions and also to study the homology to fungi PKS