Tesis sobre el tema "Isoprenoidi"

Los estudios realizados sobre las proteínas Arv indican que estaría involucrada en la regulación de la homeostasis de esteroles y esfingolípidos. En levadura se observó una posible función en la síntesis de GPI (Kajiwara et al., 2008) y respecto a su topología, posee 3 dominios hidrofóbicos con el extremo N-terminal orientado hacia el citosol (Villasmil y Nickels 2011). En plantas, se caracterizaron los genes AtARV1 y AtARV2 de Arabidopsis thaliana que codifican dos proteínas de RE funcionales, AtArv1 y AtArv2. Estas comparten una identidad del 66% entre sí y poseen un dominio N-terminal AHD muy conservado en diferentes especies. Ambos genes poseen patrones solapantes de expresión en tejidos meristemáticos, excepto en hojas donde AtARV2 no se expresa (Forés et al., 2006). En este trabajo se determinó que AtArv1 posee un número par de dominios hidrofóbicos y que los extremos N- y C-terminales están orientados hacia el citosol. Además, se caracterizaron mutantes simples con pérdida de función (arv1) y dobles (arv1:arv2), que si bien no mostraron alteraciones fenotípicas, la activación de un RNAi en los doble mutantes arv1:arv2 indica que la pérdida de AtArv produce alteraciones fenotípicas en plántulas, que presentan acortamiento de la raíz y de los cotiledones que se vuelven amarillentos y se curvan en forma de « punta de flecha ». Los niveles de esteroles disminuyen y se ven incrementadas algunas BECL (Bases Esfingoides de Cadena Larga). Por otro lado, AtArv1 no parece estar vinculada a la UPR (Unfolded Protein Response) y su pérdida no es capaz de activar esta respuesta. La farnesildifosfato sintasa (FPS) es una enzima dimérica que cataliza la condensación de una molécula de dimetilalildifosfato con dos moléculas de isopentenildifosfato para dar lugar a farnesildifosfato, que es el punto de partida para la síntesis de isoprenoides en el citosol y las mitocondrias. Los genes FPS1 y FPS2 de Arabidopsis thaliana codifican las isoenzimas FPS1L mitocondrial), FPS1S y FPS2. Las secuencias aminoacídicas de FPS1S y FPS2 poseen una identidad del 90.6% y difieren en sólo 32 aminoácidos. El gen FPS1 se expresa de forma generalizada durante todo el desarrollo de la planta, en cambio, FPS2 muestra un patrón de expresión restringido a órganos florales, semillas y estadios tempranos de la germinación. En la caracterización de los mutantes nulos de Arabidopsis fps1 y fps2 se observó que el gen FPS2 es más importante en las semillas y las etapas tempranas de la germinación. La isoenzima FPS2 contribuye entre un 70-80% a la actividad FPS total en semillas maduras. Como consecuencia, las semillas del mutante fps2 presentan niveles reducidos de sitosterol, un aumento de la actividad HMG-CoA reductasa (HMGR) e hipersensibilidad a la mevastatina (Closa et al., 2010). En este trabajo se ha determinado mediante ensayos bioquímicos, que la isoenzima FPS2 es catalíticamente más eficiente, más sensible al efecto inhibitorio del NaCl, y termodinámicamente más estable que FPS1S. También se ha demostrado la localización citosólica de ambas isoenzimas. Los patrones de expresión de los genes FPS1 y FPS2 son complementarios en semilla e indican que el FPP necesario para el desarrollo de las semillas tiene dos posibles orígenes separados en el espacio y en el tiempo. Por último, los estudios de complementación funcional en semillas del mutante fps2 demuestran que en condiciones normales FPS1S y FPS2 son funcionalmente intercambiables.
The previously characterized Arabidopsis thaliana proteins AtArv1 and AtArv2 have been suggested to be involved in the regulation of cellular lipid homeostasis as demonstrated for their yeast and mammalian counterparts. In this study, we established the citosolic orientation of both N- and C-terminal ends of the AtArv1 protein in the yeast ER membrane. Functional complementation assays of an arv1Δ yeast strain with a truncated AtArv1 protein also showed that the C-terminal 31 aminoacids are essential for AtArv1 function. Characterization of single Arabidopsis arv mutants did not reveal any effect on plant phenotype. On the contrary, characterization of loss-of-function Arabidopsis arv1:arv2 double mutants obtained by inducible siRNA-mediated silencing of AtARV genes demonstrated that lack of AtArv function leads to reduced root lenght and pale green curved cotiledons as well as to reduced levels of major sterols and increased levels of some sphingolipid LCBs (Long Chain Bases). In contrast to S. cerevisiae Arv1p, AtArv is not involved in the UPR (Unfolded Protein Response) in Arabidopsis since lack of AtArv1 does not activate this response. Previous results obtained in our laboratory showed that Arabidopsis thaliana contains two genes encoding farnesyl diphosphate (FPP) synthase (FPS), the short-chain prenyl diphoshate synthase that catalyzes the synthesis of FPP from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The FPS1 gene is widely expressed in all plant tissues throughout development, whereas FPS2 shows a pattern of expression restricted to specific floral organs, developing and mature seeds. Characterization of fps single knock-out mutants suggested that FPS2 has a major role in seeds and during the early stages of seedling development. Actually, FPS2 provides 70-80% of total FPS activity in mature Arabidopsis seeds, hence lack of FPS2 activity in seeds leads to a marked reduction in sitosterol content and a positive feedback regulatory response of HMG-CoA reductase (HMGR) activity that renders seeds hypersensitive to mevastatin. In this study, we provide evidence that the two Arabidopsis short FPS isozymes FPS1S and FPS2 localize to the cytosol. Biochemical characterization of these recombinant enzymes expressed in E. coli, revealed that, despite FPS1S and FPS2 share more than 90% amino acid sequence identity, FPS2 is more efficient as a catalyst, more sensitive to the inhibitory effect of NaCl, and more resistant to thermal inactivation than FPS1S. Expression analysis of FPS::GUS genes in seeds also showed that FPS1 and FPS2 display complementary patterns of expression particularly at late stages of seed development, which suggests that developping Arabidopsis seeds have two spatially segregated sources of FPP. Functional complementation studies of the fps2 knock-out mutant seed phenotypes demonstrated that at least under normal conditions FPS1S and FPS2 are functionally interchangeable.

Orientador: Raquel Marques Braga
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica
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Resumo: Nosso trabalho tem por objetivo o isolamento e a elucidação estrutural dos metabólitos secundários de Himatanthus obovatus (família Apocynaceae, sub-família Rauvolfioideae). Apenas cinco espécies de Himatanthus já foram estudadas do ponto de vista químico. O material de H. obovatus utilizado nesse trabalho foi coletado na Chapada dos Guimarães (MT) e em Casa Branca (SP). Utilizando diferentes metodologias de extração e tratamento dos extratos etanólicos brutos foram isoladas 5 lignanas: pinoresinol, isolariciresinol, hidroxipinoresinol, lariciresinol e olivil; 3 nor-isoprenóides: blumenol C, blumenol A e um nor-isoprenóide inédito; o iridóide plumieride, misturas dos terpenos: acetato de lupeol + acetato de a-amirina + acetato de b-amirina + germanicol e stigmasterol + b-sitosterol + campesteroI a, após a acetilação do extrato etanólico bruto, o glicitol inositol. Os extratos Diclorometânico (CDCb) e Etanólico (CECb) da casca de H. obovatus (Casa Branca (SP) foram submetidos aos testes com Artemia salina Leach. e de atividade antiproliferativa frente à 4 linhagens celulares derivadas de tumores humanos: leucemia (K562), pulmão (NCI460), melanoma (UACC62) e mama (MCF7). Os resultados dos dois testes foram bastante coerentes, já que ambos mostraram resultados promissores para o extrato CDCb. Os testes de bioautografia foram realizados com os extratos da casca de H. obovatus (Casa Branca): CHCb (heptano), CDCb (diclorometano) e CECb (etanol) e com as substâncias isoladas: pinoresinol, isolariciresinol, blumenol C, blumenol A, nor-isoprenóide inédito, hidroxipinoresinol, lariciresinol, plumieride e inositol, frente aos fungos: Alternaria alternata, Aspergillus fumigatus, A. niger, Candida albicans, Cladosporium cladosporioides, Fusarium oxysporium, Penicillium oxalicum, P. funicullosum e Rhizopus orizae. e frente às bactérias: Bacillus subtilis, Escherichia coli, Micrococcus luteus, Salmonella typhimurim, Staphilococcus aureus e Streptococcus mutans. Foram observadas atividades bactericida para as lignanas: isolariciresinol frente à bactéria S. mutans e lariciresinol frente à bactéria S. aureus. Os compostos isolados de H. obovatus permitiram comparar filogeneticamente este gênero aos gêneros Tabernaemontana e Rauvolfia (pertencentes às mesmas família e sub-família), estudados anteriormente em nosso grupo de pesquisas e ricos em alcalóides indólicos.
Abstract: Our objective is the isolation and identification of the compounds from Himatanthus obovatus (family Apocynaceae and sub-family Rauvolfioideae). Five species from genus Himatanthus have been chemically studied. H. obovatus was collected in Chapada dos Guimarães (MT state, Brazil) and in Casa Branca (SP State, Brazil). We used different methodologies for extraction and purification of the extracts, yielding 5 lignans: pinoresinol, isolariciresinol, hydroxypinoresinol, lariciresinol and olivil; 3 nor-isoprenoids: blumenol C, blumenol A and one unknown nor-isoprenoid; the iridoid plumieride, a mixture of terpenes: lupeol acetate + a-amirin acetate + b-amirin acetate + germanicol and stigmasterol + b-sitosterol + campesterol and, after acetylation of the crude ethanolic extract, the glycitol inositol. The diclorometanic (CDCb) and ethanolic (CECb) extracts trom the bark of H. obovatus (Casa Branca - SP) have been tested with Artemia salina Leach. and for antiproliferative activity against 4 carcinoma cell lines derived from human cancer: leukemia (K562), lung (NCI1460), melanoma (UACC62) and breast (MCF7). The good results with the CDCb extract in both tests suggest that this extract is a development candidate. The Bioautography tests were made with the heptanic (CHCb), diclorometanic (CDCb) and ethanolic (CECb) extracts from the bark of H. obovatus (Casa Branca - SP) and with the isolated substances: pinoresinol, isolariciresinel, blumenol C, blumenol A, the unknown nor-isoprenoid, hydroxypinoresinol, lariciresinol, plumieride and inositol against the fungi: Alternaria alternata, Aspergillus fumigatus, A. niger, Candida albicans, Cladosporium cladosporioides, Fusarium oxysporium, Penicillium oxalicum P. funicullosum and Rhizopus orizae and against the bacteria: Bacillus subtilis, Escherichia coli, Micrococcus luteus, Salmonella typhimurim, Staphilococcus aureus and Streptococcus mutans. We observed bactericide activity at the lignans: isolariciresinol against the bacteria S. mutans and lariciresinol against the bacteria S. aureus. The coumpounds isolated from H. obovatus allowed us to phylogenetically compare this genus to the genera Tabernaemontana and Rauvolfia (belonging to the same family and sub-family), previously studied in our group and rich in indolic alkaloids.
Doutorado
Quimica Organica
Doutor em Ciências

Els isoprenoides constitueixen una vasta família de compostos naturals que duen a terme una amplia varietat de funcions, moltes d’elles essencials, en els tres dominis de la vida. No obstant tots ells deriven d’una única molècula comú, el IPP (i el seu isòmer DMAPP) que es produeix a través de dues possibles rutes biosintètiques, la via del MEP i la via del MVA. La via MEP és la predominant en eubacteris mentre que la del MVA ho és en arqueobacteris, fongs i animals. En plantes coexisteixen les dues vies de forma compartimentalitzada. Aquesta distribució diferencial de les dues vies ha convertit la via MEP en una nova i prometedora diana per al disseny de nous antibiòtics (amb la FSM, un inhibidor específic de l’enzim DXR de la via MEP com a màxim exponent), molt necessaris en l’actualitat com a conseqüència de l’emergent resistència que han desenvolupat un gran nombre de microorganismes patògens a les drogues desenvolupades fins al moment. No obstant, tot i que el caràcter essencial dels isoprenoides fa que la seva síntesi es mantingui en tots els éssers vius la enorme plasticitat gènica, que es concentra especialment en bacteris, ha donat lloc a l’adopció d’estratègies alternatives tant a nivell d’enzims com metabòlits. Els carotenoides conformen una subfamília dintre dels isoprenoides de compostos derivats de la molècula parental de 40 carbonis, el fitoe, amb funcions que comprenen tant el metabolisme primari com el secundari. Es sintetitzen principalment en els plastidis de les plantes però també són produïts per alguns bacteris no fotosintètics i fongs. La seva creixent aplicació en la indústria ha fet molt atractiva l’aplicació de la biotecnologia per a la producció d’aquests compostos però per poder afrontar amb garanties aquest repte cal conèixer a fons els mecanismes que regulen a tots nivells la seva producció. Al llarg d’aquest treball s’ha combinat l’estudi dels aspectes bàsics i aplicats de la síntesi d’isoprenoides amb especial èmfasi en el paper de la via del MEP i la biosíntesi de carotenoides per proposar i avaluar estratègies de millora de la seva producció tant en bacteris com en plantes. El treball ha permès testar l’us d’enzims bifuncionals per millorar simultaniàment dos passos catalítics de la ruta carotenogènica tant en sistemes bacterians com en plantes. Es va analitzar l’efecte de la fusió de LCYE i LCYB (ε-ciclasa i β-ciclasa respectivament) en expressar-la en un sistema model carotenogènic de E.coli o en plantes transgèniques d’Arabidopsis thaliana obtenint-se resultats positius en el primer cas però no en el segon. Aquesta diferència posa de manifest la major complexitat dels sistemes vegetals. En aquesta línia es va explorar el possible paper addicional de la regulació post-transcripcional en el control del flux de la via del MEP utilitzant un mutant (rif18) obtingut en base a un escrutini de resistència a la inhibició per FSM. L’estudi va posar de manifest un nivell de regulació com a resultat de la integració i coordinació de la ruta MEP amb la resta del metabolisme de la planta especialment els metabolismes del sucre i de les hormones. Paral.lelament es va avaluar el paper que juguen individualment cada un dels enzims de la via MEP en el control del flux de la via en sistemes bacterians. L’estudi de la sobreexpressió de cada un dels gens en un sistema model carotenogènic de E.coli va posar de manifest el paper clau de DXS i la participació en menor mesura de DXR i HDR, resultats similars als descrits en Arabidopsis. Però, encara més important, va demostrar que uns nivells moderats de sobrexpressió dels enzims clau produeixen millors resultats en l’acumulació de productes finals. Complementariàment es va estudiar en profunditat el paper de HDS tant en bacteris com en plantes ja que havia estat descrit préviament com un possible punt de regulació diferencial entre els dos sistemes en base a les diferències observades en alineaments de seqüència. En cap dels dos casos un increment de l’activitat d’aquest enzim millorava l’acumulació de productes finals. Els dos passos identificats com a claus en la ruta MEP (DXS i DXR) es van escollir com a objectius sobre els que, aprofitant la plasticitat gènica i metabòlica dels bacteris, cercar noves activitats alternatives. Per DXS es van identificar dues proteïnes de E.coli (E1-PDH i DHBPS) que en patir mutacions puntuals adquirien la capacitat de complementar la deficiència de DXS. Per identificar activitats alternatives a DXR es va analitzar la presència d’homòlegs pels 7 gens de la via MEP en els genomes seqüenciats de microorganismes. Es va identificar un grup en els que hi eren presents tots ells excepte DXR. Utilitzant el genoma de Brucella abortus es va identificar una proteïna sense homologia amb DXR (DRL) capaç de suplir-ne l’absència i que defineix una nova família de proteïnes la funció de les quals era desconeguda. La seva caracterització va mostrar que catalitza la mateixa reacció amb propietats cinètiques similars a les descrites per DXR però amb diferències significatives, com una menor eficiència catalítica o una menor sensibilitat a la inhibició per FSM. L’estudi filogènetic de la família va posar de manifest l’existència de DRL vertaderes (amb activitat DXR) que s’agrupaven en un mateix clade filogenètic i DRL falses definint a l’hora tres classes de proteïnes. La cristal.lització i resolució de l’estructura tridimensional de la proteïna DRL de Brucella abortus va permetre un estudi estructural comparatiu observant-se diferències significatives respecte DXR especialment en el reordenament del centre actiu. A partir d’aquestes diferències es va hipotetitzar, en base a modelitzacions in silico, la possibilitat d’inhibir diferencialment els dos enzims. Aquesta possibilitat es va confirmar en estudis d’inhibició in vitro amb les proteïnes DXR de E.coli, i DRL de B.abortus recombinants purificades. Els α-fenil derivats de la FSM mostraven una capacitat d’inhibició de DXR en l’ordre nanomolar mentre que no mostraven cap efecte sobre DRL a concentracions de fins a 1 mM obrint la porta al disseny de nous antibiòtics de gran especificitat.
ISOPRENOIDS BIOSYNTHESIS IN BACTERIA AND PLANTS Isoprenoids are a vast family of natural compounds that perform a wide variety of biological functions, many of which are essential. However, they all derived from the universal intermediates IPP and its isomer DMAPP. Their biosynthesis occurs through two possible biosynthetic routes, the MEP and the MVA pathways. The MEP pathway is the exclusive source of IPP and DMAPP in eubacteria whereas the MVA pathway provides these precursors in archaea, fungi and animals. In plants, both pathways coexist in different subcelullar compartments. This distribution of isoprenoid biosynthetic pathways among the kingdoms has turned the MEP pathway into a promising new target for the design of new antibiotics (with FSM, a specific inhibitor of the enzyme DXR, as the main representative). Although isoprenoids are essential in all free-living organisms, tremendous plasticity among bacteria has led to the evolution of alternative biochemical strategies to produce these universal precursors. Carotenoids comprise a subfamily of C40 isoprenoid end products derived from a molecule of phytoene with functions in both primary and secondary metabolism. These are synthesized primarily in the plastids of plants but are also produced by some photosynthetic bacteria, fungi, and rarely in insects. The increasing use of carotenoids in the food, cosmetics and pharmaceutical industries as pigments, fragrances and nutraceuticals has galvanized their production through biotechnological applications. We have combined the study of basic and applied aspects of isoprenoid biosynthesis focusing on the role of the MEP pathway and carotenoid biosynthesis to suggest and evaluate strategies for improving their production in bacteria and plants. To this end, we have exploited the metabolic plasticity of bacteria to provide new biotechnological tools for carotenoid production. In the study, the use of bifunctional enzymes was tested to simultaneously improve two catalytic steps. As a proof of concept we analyzed the effect of chimeric LCYE-LCYB (ε-cyclase and β-cyclase, respectively) in a carotenogenic E. coli model system or in transgenic plants of Arabidopsis thaliana. The difference in the results highlights the major complexity of plant systems. In this direction, we studied the possible role of additional post-transcriptional regulation controlling the flow of the MEP pathway by using a mutant (rif18) isolated from a FSM-resistance screening. The study showed a higher level of regulation as a consequence of the integration and coordination with other metabolic pathways of the plant, especially the metabolism of sugars and hormones. We also evaluated the role of each of the individual enzymes of the MEP pathway in controlling the pathway flux in bacterial systems. Overexpresion of each individual gene in a carotenogenic E. coli model system highlighted the key role of DXS and to a lesser extent of the participation for DXR and HDR in driving flux through this pathway, in agreement with previous reports for Arabidopsis. Even more importantly, these results demonstrate that adjusted levels of overexpresion of the key enzymes produce better results in the accumulation of end products, the most relevant parameter for evaluating biotechnological strategies. Additionally, we further studied the role of HDS both in bacteria and plants. This enzyme had been previously postulated to have different regulatory roles in the two systems based on protein alignments. In both cases, an increase in enzyme activity does not affect the accumulation of end products. The identified key steps in the MEP pathway (DXS and DXR) were chosen as targets to search for new alternative activities taking advantage of the genetic and metabolic plasticity of bacteria. Two E.coli proteins which underwent change-of-function mutations in dxs or dxr deficient strains and acquired DXS activity were identified (DHBPS and PDH-E1). An alternative activity to DXR was identified by analyzing the sequenced genomes available from bacteria. A small group of microorganism contains homologous genes for all MEP pathway but DXR. Using the genome of Brucella abortus, a separate protein with no apparent homology to DXR (thereafter named DXR-like, or DRL) was identified. DRL defines a new family of proteins that catalyzes the first committed step of the MEP pathway. Characterization of the protein showed that it catalyzes the same reaction with similar kinetic properties as DXR albeit with significant differences in catalytic efficiency and lower sensitivity to inhibition by FSM. Phylogenetic and complementation studies of the family revealed the existence of true DRL (with DXR activity) grouped in the same phylogenetic clade. Crystallization and structural resolution of DRL from Brucella abortus allowed a comparative study. Significant differences to DXR were observed, in particular in the arrangement of the active site. Based on those differences and in silico modeling, the possibility to selectively inhibit either enzyme was hypothesized. In vitro inhibition studies using purified recombinant E. coli DXR and B.abortus DRL demonstrates that α-phenyl derivatives of FSM inhibit DXR in the nanomolar range while show no significant effect on DRL at a concentrations up to 1 mM. Those results open the door for the design of new highly specific antibiotics. The first tests in biotechnological applications comparing the ability of the three alternative proteins to replace the original DXS or DXR activities showed no improvement in performance. However, these proteins should be considered starting materials to continue the optimization of the new catalytic functions acquired using directed enzyme evolution methods in the laboratory.

This thesis describes a possible alternative isoprenoid pathway in bacteria by considering some previously unpublished feeding studies in the context of the related background literature. Three synthetic routes to 2,4-dihydroxy-4-methyltetrahydropyran (63) and three synthetic strategies towards the synthesis of 2-carboxy-2,4-dihydroxy-4-methyltetrahydropyran (63) are discussed. These compounds are considered as potential intermediates in the proposed alternative bacterial isoprenoid pathway. Labelled synthesis of (63) and structural analysis of (63) and 4-hydroxy-2-methoxy-4-methyltetrahydropyran (99) by proton nmr are also described. Feeding studies including the ¹³C isotopically labelled tetrahydropyrans (63) and (99) are described and a revised interpretation of all of the feeding studies considered. HMGCoA synthase is assayed in Rh. capsulata after a description of its assay in bakers yeast.

Parkinson???s disease (PD) is a progressive neurodegenerative disease characterised pathologically by the selective death of the dopaminergic neurons of the substantia nigra and the appearance of abnormal inclusions in some surviving neurons. A body of evidence from epidemiological, in vitro and in vivo studies suggest that isoprenoids, a lipid family which includes cholesterol, dolichol and ubiquinone, may play a role in PD, although to date the data has been conflicting with little consensus regarding the type or direction of change in isoprenoids in PD. The current study investigated isoprenoids in PD by quantifying a range of isoprenoids in blood sera, brain homogenates and olfactory mucosa derived from PD patients and controls. Further, isoprenoid synthesis pathways were investigated by comparing the activitites and amount of the rate-limiting enzyme for isoprenoid synthesis, HMG CoA reductase, in olfactory mucosal cultures from individuals with sporadic PD and leucine-rich repeat kinase 2 (LRRK2)-PD with those from healthy individuals. Serum levels of total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides and dolichol were unchanged in patients with PD compared with controls. Similarly, total tissue cholesterol was unchanged in degenerating and non-degenerating regions of the PD brain, but tissue dolichol was significantly decreased in the substantia nigra in the PD brain, possibly reflecting a change in the neuron/glia ratio in this brain region. In olfactory mucosa, a significant decrease in cellular cholesterol content was identified in patients with LRRK2-PD compared with patients with sporadic PD or controls. The reduction in cholesterol was similar in two different LRRK2 mutations but was not associated with a change in either the amount or activity of HMG CoA reductase. This study suggests that decreased cholesterol is associated with LRRK2-PD but not with sporadic PD. As cholesterol levels in cells with different LRRK2 mutations were reduced to a similar extent, it is suggested that mutations in this gene result in a loss-of-function of LRRK2 protein. Further it suggests a role for LRRK2 in cholesterol homeostasis independent of HMG-CoA reductase-associated pathways. Recent data has suggested a functional role of LRRK2 in autophagy, a mechanism which may explain the reduction in cholesterol observed in LRRK2-PD.

Il etait unanimement accepte que la biosynthese des isoprenoides passait par le mevalonate. Des etudes recentes ont revele l'existence d'une voie alternative independante du mevalonate passante par le glyceraldehyde 3-phosphate (gap) et la pyruvate via une condensation et un rearrangement. Nous avons etudie le role du 2-c-methyleerythritol comme precurseur dans cette voie. En effet, des incorporations de glucose marque au #1#3c ont montre que chez corynebacterium ammoniagenes les squelettes du methylerythritol et des unites isopreniques etait formes de la meme facon. L'incorporation de #2h-2-c-methyl-d-erythritol dans les isoprenoides d'escherichia coli a confirme le role de ce polyol ramifie comme precurseur de l'ipp. La bacterie zymomonas mobilis produit des hopanoides complexes. La conservation des deux atomes de deuterium sur le carbone c-31 apres incorporation du 6,6-#2h#2glucose indique qu'une reaction de substitution permet l'insertion de la chaine laterale en c#5. Les marquages de la squelette hopane a confirme l'intervention de la voie gap/pyruvate. Nous avons developpe une hemisynthese du ribosylhopane qui donne acces a des hopanoides qui pourrait etre des precurseurs des bacteriohopanepolyols ou des hopanoides avec des activites biologiques interessantes. Nous avons synthetise un analogue du bacteriohopanetetrol en serie sterane avec une chaine laterale c#5 polyhydroxylee lie au carbone c-3 en utilisant une strategie synthetique similaire a celle utilisee pour l'obtention du ribosylhopane. Cet analogue a ete propose comme precurseur biologique des 3-alkylsteranes et des 3-carboxyalkylsteranes, abondants dans les sediments et serait a rechercher dans des microorganismes.

Products of the isoprenoid biosynthetic pathway are involved in diverse biological functions. For example, the isoprenoid diphosphate, farnesyl diphosphate (FPP), is used for synthesis of squalene, a precursor of cholesterol. In addition, FPP and geranylgeranyl diphosphate (GGPP) are used for protein prenylation, which is a post-translational modification of certain proteins required for their proper membrane localization and function. Enzymes within the isoprenoid biosynthetic pathway have been inhibited successfully by drugs that are now used clinically, including statins and nitrogenous bisphosphonates (NBPs). Statins and NBPs are inhibitors of isoprenoid biosynthetic enzymes, due to their structural resemblance to substrates within the pathway. The bisphosphonate core resembles the diphosphate portion of isoprenoid diphosphate intermediates within the isoprenoid biosynthetic pathway. It is hypothesized that distinct isoprenoid biosynthetic enzymes can be inhibited by bisphosphonates in a manner dependent upon the overall bisphosphonate structure. Along with our collaborators, we have developed novel bisphosphonate inhibitors of multiple isoprenoid biosynthetic enzymes. Potent in vitro inhibitors of squalene synthase (SQS) were identified and evaluated in HepG2 liver cells. A lead inhibitor of squalene synthase was combined with a statin and a nitrogenous bisphosphonate, and focus was placed on these combinations as potential novel mechanisms to reduce cholesterol synthesis while minimizing impairment of non-sterol synthesis. Specifically, it was found that the lead SQS inhibitor prevents lovastatin-mediated impairment of protein farnesylation but not geranylgeranylation. Also, the lead SQS inhibitor prevented both zoledronate-induced impairment of protein farnesylation and geranylgeranylation. Novel bisphosphonates were also identified as inhibitors of geranylgeranyl diphosphate synthase (GGDPS) and protein prenylation in K562 leukemia cells. A novel cellular consequence of GGPP depletion was also established. In PC3 cells, zoledronate and digeranyl bisphosphonate (DGBP; a lead inhibitor of GGDPS) were determined to induce autophagy as measured by accumulation of the autophagic marker LC3-II. GGPP depletion was implicated as the cause of autophagic induction in this system. Specifically, results suggest that impairment of proteins geranylgeranylated by geranylgeranyl transferase II is responsible for the induction of autophagy. Mycobacterium isoprenoid biosynthetic enzymes were also evaluated as inhibitory targets for bisphosphonates. Novel inhibitors of Mycobacteria tuberculosis omega-E,Z-FPP synthase and decaprenyl diphosphate synthase were identified. A lead inhibitor of decaprenyl diphosphate synthase was also evaluated in Mycobacterium smegmatis, which was utilized as a surrogate model. The lead inhibitor was found to have no effect on M. smegmatis growth; however it enhanced growth inhibition mediated by ethambutol. This effect was prevented by addition of exogenous decaprenyl diphosphate, suggesting that the growth inhibition was due to decaprenyl diphosphate depletion. Decaprenyl diphosphate was also found to prevent the growth inhibitory effect of SQ109, a novel anti-mycobacterial drug in clinical development with an unknown mechanism of action.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2019
Cataloged from PDF version of thesis.
Includes bibliographical references.
Isoprenoids comprise a large class of chemicals, of significant interest due to their diverse properties. Most isoprenoids are plant secondary metabolites and are of commercial importance due to their varied applications in fields spanning medicine, agriculture, flavors, fragrances, cosmetics and nutrition. Biological production of isoprenoids in microbes is considered to be the most efficient and commercially viable way for their large-scale production. Thus far, isoprenoid biosynthesis has been performed through pathways inextricably linked to glycolysis. Furthermore, these pathways are inherently limited due to their extensive cofactor requirements, complex regulation and large number of steps. In this thesis we present a novel pathway for isoprenoid synthesis, the Isopentenol Utilization Pathway (IUP), which aims to overcome these limitations.
This pathway functions through the double phosphorylation of an isopentenol, either isoprenol or prenol, to produce the main precursors to isoprenoid synthesis, isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). This pathway is radically different from naturally-occurring pathways or their engineered variants because it is only two steps long, uses an externally-provided isoprenol as its substrate instead of a glucose-derived catabolite, and uses only a single co-factor, ATP. We identify suitable enzymes, construct the pathway and proceed to demonstrate an in vivo proof of concept. After optimizing the pathway feedstock, we proceed to show that IUP is decoupled from central carbon metabolism. We demonstrate that the IUP can quickly produce copious amounts of IPP & DMAPP and can be used for the production of a variety of isoprenoids.
The IUP flux exceeded the capacity of almost all downstream pathways tested, was competitive with the highest isoprenoid fluxes reported as well as against state-of-the art isoprenoid pathways. Furthermore, we elaborate on our progress towards improving the capacity of a downstream farnesene synthesis pathway, to catch up with and fully utilize IUP's production capacity. Finally, we propose a new scheme for the use of the IUP to produce functionalized isoprenoids using functionalized isopentenols to introduce functionalizations in isoprenoid backbones, and we show preliminary results of this application.
by Alkiviadis Orfefs Chatzivasileiou.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Chemical Engineering

Les lipides, et en particulier les isoprenoides, presents dans la matiere organique des sediments, sont souvent utilises comme biomarqueurs pour reconstituer des paleoenvironnements. Pour ameliorer la comprehension de ces biomarqueurs, nous avons analyse les triterpenoides d'organismes qui peuvent contribuer de facon significative a cette matiere organique. Ce criblage nous a permis de detecter de nouveaux hopanoides insatures chez les bacteries burkholderia et methylocaldum spp. , et de confirmer que les derives du gammacerane et du 17h,21h hopanes sont repandus chez les eubacteries. De plus, nous avons montre que les lipides des rotiferes proviennent directement de leur diete sans modification et qu'ils representent une empreinte moyenne du plancton de leur environnement. Nos analyses sont en accord avec le fait que les crustaces planctoniques modifient les lipides, en particulier desalkylent les phytosterols, supprimant ainsi la majeure partie des informations provenant du phytoplancton. Nous avons ainsi pu montrer que les triterpenoides du plancton de six lacs europeens se retrouvent dans l'empreinte des biomarqueurs trouves dans les sediments recents de ces lacs, et reciproquement que l'analyse des sediments d'un lac peut fournir des indication sur les principaux contributeurs a la matiere organique de ce systeme. Nous avons egalement effectue des experiences d'incorporations de precurseurs marques au 1 3c et au 2h sur des algues unicellulaires, constituants de phytoplancton, a savoir la diatomee phaeodactylum tricornutum et le dinoflagelle glenodinium foliaceum, afin de determiner la voie de biosynthese des isoprenoides chez ces organismes. Les resultats montrent la meme compartimentation de la biosynthese chez ces algues que chez les plantes : les isoprenoides situes dans les chloroplastes sont formes via le methylerythritol phosphate, et ceux dans le cytoplasme via le mevalonate. Ces experiences ont ete effectuees dans des conditions mixotrophiques. Nous avons egalement tenter de differencier les deux voies de biosynthese des isoprenoides chez les organismes phototrophes par le rapport isotopique 1 3c/ 1 2c naturel, dans les conditions naturelles de la croissance de ces organismes. Pour le moment, cette differentiation n'est pas possible, et une application eventuelle de rapport isotopique dans ce domaine exige des etudes approfondies.

Isoprenoids are a class of secondary metabolites that are widely distributed in Nature. This thesis describes the synthesis and feeding of isotopically labelled enriched substrates to elucidate features of a new mevalonate independent pathway to isoprenoids. Chapter 2 describes studies with a whole plant system, Mentha citrata, which produces the monoterpene linalyl acetate, and a bacterium Escherichia coli which produces ubiquinone-8. Feeding experiments with stable isotopically enriched compounds demonstrate that the terpene unit of linalyl acetate is biosynthesised via the mevalonate independent pathway, hicorporation of deuterium from [6,6-(^2)H(_2)]-glucose, [(^2)H(_3)]-alanine and [(^13)C(^2)H(_3)]-alanine into luialyl acetate show that the conversion to isopentenyl pyrophosphate does not proceed via a series of dehydrations. Feeding experiments with putative intermediates bearing deuterium into E. coli show that none of the intermediates are incorporated. This suggests that E. coli lacks a kinase to activate exogenously administered substrates fed as the free alcohols. Chapter 3 outlines biosynthesis studies on a meroterpene rosnecatone produced by the fungus R. necatrix. Intact incorporation of (^13)C from the feeding of [l,2-(^13)C(_2)]-acetate shows that the terpenoid moiety is produced via the mevalonic acid pathway and the non-terpenoid unit is polyketide derived. Incorporation of deuterium from [6,6-(^2)H(_2)]-glucose fully describes the pentaketide that delivers the non-terpenoid fragment. The effect on the metabolite production of changing the growth conditions of R. necatrix is investigated. Changing from a static culture to a submerged cultures causes an increased rate of growth, an upregulation of the production of cytochalasan E and a cessation of rosnecatone production. Screening of rosnecatone against two Human cancer cell lines shows IC(_50) values of 4.48µM and 5.78µM.

Phosphonates represent an important class of organophosphorus compounds. Their use as reagents in organic synthesis is prevalent, and there is a plethora of examples of biologically active compounds possessing the phosphonate moiety. To further our exploration of phosphonates as both reagents and biologically active compounds we have developed a one-flask protocol for the direct synthesis of phosphonates from benzylic and allylic alcohols. This transformation is unprecedented and is applicable to a range of substrates. Both electron rich and electron deficient benzylic alcohols react under the conditions developed. Furthermore, good yields are achieved when converting allylic alcohols to the corresponding allylic phosphonates. In at least one case, the one-flask protocol allows for phosphonate formation that was not achievable under the standard conditions. Bisphosphonates represent a significant subclass of phosphonates. Several nitrogenous bisphosphonates have found use in the clinic as treatments for bone-related disease including osteoporosis, and there is speculation that bisphosphonates that are enzyme-specific inhibitors may be used as cancer therapies. To develop our understanding of isoprenoid metabolism, we have prepared a range of bisphosphonates as potential inhibitors of geranylgeranyl pyrophosphate synthase. After much experimentation, an α-amino analog of a potent inhibitor of GGDPS has been synthesized and biological data is forthcoming. Furthermore, a new class of aromatic bisphosphonates, analogs of digeranyl bisphosphonic acid, has been synthesized and assayed. The bioassay results indicate that this series of compounds retains its specificity for the GGDPS enzyme, and that the dialkyl analogues retain much of their potency in the assays in spite of the increased steric bulk of the aromatic substructure. We have also begun the design and synthesis of compounds as potential inhibitors of Rab geranylgeranyl transferase (RGGTase). The lead compound, 3-PEHPC, is documented to inhibit RGGTase selectively, albeit at less than desirable concentrations. Using 3-PEHPC as the model compound we have elected to probe the impact of modifications on the hydrophilic "head" portion of the molecule. Using the phosphonophosphinate functionality as a surrogate for the phosphonocarboxylate moiety we have successfully synthesized digeranyl phosphonophosphinate. Initial assay data indicates that this novel phosphonophosphinate does not act upon GGDPS as does the analogous bisphosphonate substructure. The bioassay data to probe this compound's impact on RGGTase is forthcoming. Given the worldwide impact of tuberculosis infection and the emergence of drug-resistant strains of tuberculosis-causing pathogens, new and potent treatments for tuberculosis are necessary. We have engaged in the synthesis of several compounds as inhibitors of Rv2361c, an enzyme key to cell wall biosynthesis in Mycobacterium tuberculosis, the principle causative agent of tuberculosis in humans. To probe the impact of modifications at the C-9-position of the most potent of our Rv2361c inhibitors, we have made several analogues having phenyl and indole substituents. The in vitro enzyme assay data for the set of compounds has clarified understanding of the essential components of the pharmacophore, and helped to establish the direction for future efforts.

La enzima FPS ocupa una posición clave en la vía del MVA, ya que a partir de sus sustratos, IPP y DAMPP, y del producto de la reacción que cataliza, FPP, se sintetizan compuestos isoprenoides imprescindibles para el crecimiento y desarrollo de las plantas, y para su relación con el entorno. A. thaliana posee dos genes FPS, AtFPS1 y AtFPS2, que codifican tres isoenzimas, FPS1L, FPS1S y FPS2. La caracterización de mutantes de A. thaliana con pérdida de función de los genes AtFPS1 y AtFPS2 demostró que la actividad FPS es esencial para el desarrollo de las plantas, ya que el doble mutante Atfps1:Atfps2 presenta letalidad embrionaria. La letalidad embrionaria del doble mutante hizo imposible estudiar en profundidad la función y los mecanismos de regulación en los que se encuentran implicadas las isoenzimas FPS en las plantas adultas. En el Capítulo de esta tesis doctoral se atenuó la expresión de los genes AtFPS empleando una estrategia de silenciamiento inducible basada en el uso de microRNAs artificiales (amiRNA). Se obtuvieron líneas mutantes de fps condicionales en donde se observó una importante reducción en los niveles de mRNA, proteína y actividad FPS, un fenotipo de clorosis y una reducción importante del tamaño especialmente en la raíz, una reducción significativa en los niveles de los esteroles mayoritarios, un aumento de la actividad HMGR, una disminución en el número, el tamaño de los cloroplastos, en conjunto con una dramática alteración en la organización de los cloroplastos y una reducción en los niveles de clorofilas y carotenoides. Además el estudio de la expresión génica global mediante RNA‐seq identificó importantes grupos de genes desregulados asociados a la homeostasis de Fe, al metabolismos antioxidante GST y a la vía de señalización del JA. Todos estos antecedentes sugieren que el bloqueo en la síntesis de FPP, además de la reducción de los niveles de isoprenoides citosólico, desencadena a través de mecanismos aún desconocidos, una respuesta que interconectan la vía de isoprenoides, ubicada en el citosol, con el cloroplasto. Este trabajo representa la primera aproximación transcriptómica sobre la función de los genes AtFPS, la cual entrega datos relevantes acerca de su efecto sobre la expresión génica global y por lo tanto, sobre su interacción en otros procesos esenciales para la planta hasta completamente desconocidos. Por otro lado, en el Capítulo II se abordó una serie de estrategias para la producción en el citosol de nerolidol, componente de la HIVPs considerado uno de los volátiles de interés ecológico y económico, por su capacidad para atraer insectos carnívoros. Trabajos previos apuntaban a que la limitante para incrementar la producción de este compuesto en el citosol atendía a la disponibilidad del sustrato FPP. Esto puede explicar el bajo rendimiento obtenido en diversos intentos realizados para producir sesquiterpenos en el citosol mediante la expresión de diferentes sesquiterpeno sintasas. En cambio, la expresión de sesquiterpenos sintasas en otros compartimientos celulares, como el cloroplasto y la mitocondria, ha permitido superar los mecanismos homeostáticos que limitan la disponibilidad del FPP citosólico, a pesar de que las sesquiterpeno sintasas son enzimas que habitualmente se encuentran en el citosol. En este contexto, mediante la expresión transitoria en hojas de N. benthamiana de las diferentes versiones de la sesquiterpeno sintasa FaNES1 anclada a las membranas del RE, se observó un incremento de la producción de nerolidol en el compartimento citosólico. Además se utilizaron enzimas bifuncionales FPS1/FaNES1 que permitieron la producción de nerolidol en el citosol. Todos estos resultados sugieren que la síntesis de nerolidol depende esencialmente de los niveles de proteína FaNES1, lo que indica que la limitación para la producción de sesquiterpenos en este compartimento es un problema de estabilidad de la proteína y no de disponibilidad de sustrato.
"Study of gene silencing synthase and farnesyl Metabolic engineering for production of sesquiterpenes in Arabidopsis" In the first chapter of this thesis, the expression of AtFPS genes was attenuated using an inducible silencing strategy based on the use of artificial microRNAs (amiRNA). fps conditional mutant lines showed a significant reduction in the levels of mRNA, protein and FPS activity. Silenced plants displayed a chlorotic phenotype and a significant reduction in the size especially in the root. A significant reduction in the levels of the major sterols was observed unless HMGR activity was significantly induced. Mutant plants showed a reduction in chloroplasts number and size which in conjunction resulted in a dramatic alteration in chloroplasts organization and a reduction of chlorophylls and carotenoids levels. Furthermore, the study of global gene expression by RNA‐seq led us to the identification of groups of deregulated genes involved in Fe homeostasis, GST metabolism and JA signaling pathway. Our data suggests that blocking the synthesis of FPP, through yet unknown mechanisms, generates a response that connect isoprenoid production in both the cytosol and the chloroplast. In Chapter II we have developed several strategies for the production of nerolidol in the cytosol, a typical sesquiterpene produced by HIPVs. We have achieved an increased production of nerolidol in the cytosol when overexpressing different versions of FaNES1 sesquiterpene synthase anchored to the ER membranes in N. benthamiana leaves. Furthermore, FPS1/FaNES1 bifunctional enzyme also enabled the efficiently production of nerolidol in the cytosol. Both FaNES1 anchored in the ER membranes as well as the cytosolic expression of a fusion protein with GFP or FPS1S isoform tremendously increase the enzyme stability and gave rise to the production of nerolidol. Our results indicate that the production of nerolidol in the cytosol essentially depends on FaNES1 protein levels, suggesting that the limitation for the production of sesquiterpenes in this compartment is a problem of stability of the protein rather than substrate availability.

Los isoprenoides son una de las mayores familias de compuestos descritos en la naturaleza. Estos compuestos están presentes en todos los organismos vivos y se sintetizan a partir de dos moléculas de 5 átomos de carbono: el isopentenil difosfato (IPP) y el dimetilalil difosfato (DMAPP). Actualmente se conoce que arqueobacterias, hongos y animales presentan la ruta del mevalonato de síntesis de estos precursores, mientras que eubacterias, algún protozoo (como el causante de la malaria) y protistas presentan la ruta del metileritritol 4-fosfato (MEP) de síntesis de IPP y DMAPP. Estas rutas coexisten separadas espacialmente en plantas, helechos y algunas algas. La ruta del MEP de biosíntesis de los precursores de isoprenoides se muestra como una atractiva diana para la búsqueda de nuevos compuestos antimaláricos, antibióticos y herbicidas debido a su presencia en los principales agentes patogénicos y su ausencia en animales, además del carácter esencial de los isoprenoides para la vida. En esta tesis se ha realizado la búsqueda asistida por ordenador de compuestos que puedan interferir en la formación del complejo homodimérico del cuarto paso enzimático de la ruta del MEP. La metodología utilizada es muy útil en la búsqueda de inhibidores específicos. Se han caracterizado la unión de diferentes compuestos obtenidos con la enzima. Además se ha caracterizado el estado de oligomerización de la enzima. Paralelamente también se ha caracterizado un homólogo del primer paso enzimático de la ruta del MEP de un organismo termofílico caracterizando sus principales parámetros cinéticos y residuos importantes para la actividad enzimática mediante mutagénesis dirigida. Como último punto, se ha caracterizado el proceso de proteólisis de diferentes homólogos de este primer paso enzimático de la ruta del MEP asociándolo a modificaciones postraduccionales intramoleculares de las mismas proteínas, abriendo la posibilidad de un proceso de regulación posttraduccional de la actividad enzimática en este tipo de enzimas.

Synthèse d’inhibiteurs potentiels non phosphorylés de la désoxyxylulose phosphate réductoisomérase et étude de la voie de biosynthèse des unités isopréniques chez Acanthamoeba polyphaga. De nombreux microorganismes pathogènes utilisent la voie du méthylérythritol phosphate (MEP) pour la synthèse des unités isopréniques (IPP et DMAPP). Absente chez l’homme, cette voie constitue une cible de choix pour lutter contre ces microorganismes. La fosmidomycine est un des meilleurs inhibiteurs connus à ce jour de la désoxyxylulose phosphate réductoisomérase (DXR), deuxième enzyme de cette voie. Afin d’améliorer d’une part la biodisponibilité de ce type d’inhibiteur et d’autre part de lutter contre le phénomène de résistance à cet antibiotique, nous avons synthétisé des analogues de la fosmidomycine où le groupement phosphonate est remplacé par un groupement tétrazole ou par un groupement squaryle. Les molécules synthétisées ont été testées sur la DXR d’ Escherichia coli et n’ont pas révélé d’activité inhibitrice significative.Par ailleurs, nous avons montré, par des expériences d’incorporation de glucose marqué au 13C, que l’amibe Acanthamoeba polyphaga, utilise la voie du mévalonate pour synthétiser les unités isopréniques nécessaires à la synthèse de ses stérols
Synthesis of potential non-phosphonate inhibitors of the deoxyxylulose phosphate reductoisomerase and study of the biosynthetic pathway for isoprenoids units synthesis using by Acanthamoeba polyphagaMany pathogenic microorganisms synthesize their isoprenoid units (IPP and DMAPP) via the methylerythritol phosphate pathway (MEP pathway). Absent in man, all enzymes of this metabolic route are potential targets for the design of new antimicrobials. This pathway is present in pathogenic bacteria, but absent in mammals. Hence, the development of small-molecule inhibitors for the MEP enzymes constitutes a novel approach for the design of new antimicrobials. Fosmidomycin is the most efficient inhibitor of the the deoxyxylulose phosphate reductoisomerase (DXR), the second enzyme of the MEP pathway. In an attempt to improve the pharmacological properties and the bioavailability of this antibiotic, we synthesized analogues of the fosmidomycin by replacing the phosphonate group by tetrazole or squaryl moieties. These synthesized compounds were tested on the DXR isolated from Escherichia coli.Otherwise, we showed by achieving incorporation experiments with 13C labeled glucose that the amoeba Acanthamoeba polyphaga utilize the mevalonate pathway to synthesize its sterols

Aquesta tesi es centra principalment en l'anàlisi funcional i la caracterització dels gens que codifiquen per a alguns metabòlits secundaris i en l’estudi de la seva regulació en les plantes. Els objectius generals varen ser (a) entendre millor la regulació transcripcional del gen de la biosíntesi dels carotenoids, la β-carotè hidroxilasa 2 (BCH2) en el blat de moro i (b) l'anàlisi funcional de les dues isopentenil difosfat isomerasas (OsIPPI) d'arròs i determinar la seva localització subcel·lular. Simultàniament, es va estudiar com la llum afecta la via metabòlica i a la producció de pelargonidina en l'arròs; es van identificar també els gens essencials de la seva biosíntesi en Gentiana lutea L. var. aurantiaca. Les plantes de blat de moro i arròs es varen transformar amb els gens dels factors de transcripció ZmMYB i ZmPBF. Es va analitzar l’expressió gènica transitòria i es va realitzar transformació estable. Els resultats obtinguts indiquen que tant ZmPBF com ZmGAMYB poden transactivar l'expressió de ZmBCH2 a l’endosperm del blat de moro, i ZmPBF i ZmGAMYB transactiven independentment el promotor de ZmBCH2 en arròs. Els dos paràlegs de IPPI (OsIPPI1 i OsIPPI2) aïllats prèviament en arròs varen tenir un patró d'expressió diferent; l'ARNm de OsIPPI1 va ser més abundant que l'ARNm de OsIPPI2 en tots els teixits. Es va usar la microscòpia de fluorescència confocal i microscòpia inmunoelectrónica per determinar la localització de les dues proteïnes. Aquestes es localitzen en el reticle endoplasmàtic (RE), així com en els peroxisomes i les mitocòndries, mentre que només es va detectar OsIPPI2 en els plastidis. La detecció d'ambdues isoformes en el RE indica que DMAPP es pot sintetitzar de novo en aquest compartiment. Diferents tècniques com UPLC, GC-MS i qRT-PCR també es varen utilitzar per perfilar els metabòlits primaris i secundaris i l'expressió gènica relacionada en plàntules d'arròs des-etioladas. Els resultats varen revelar que el metabolisme primari i secundari i els gens corresponents estan regulats per la llum, especialment en la biosíntesi d'isoprenoides en fulles d'arròs. Onze derivats de pelargonidina es varen identificar en els pètals de G. lutea i es varen perfilar els gens de la seva via de biosíntesi, revelant que DFR, ANS i 3GT afecten principalment a l'acumulació dels glucòsids de pelargonidina. Tots aquests resultats suggereixen la idea que la biosíntesi dels carotenoids en plantes superiors és regulada a diferents nivells.
Esta tesis se centra principalmente en el análisis funcional y en la caracterización de los genes que codifican para algunos metabolitos secundarios y en el estudio de su regulación en las plantas. Los objetivos generales fueron (a) profundizar en el conocimiento de la regulación transcripcional del gen de la biosíntesis de los carotenoides, la β-caroteno hidroxilasa 2 (BCH2) en el maíz, y (b) analizar la función de las dos isopentenil difosfato isomerasas (OsIPPI) de arroz, determinando además su localización subcelular. Simultáneamente, se estudió cómo la luz afecta a la vía metabólica y a la producción de pelargonidina en el arroz; se identificaron también los genes esenciales de su biosíntesis en Gentiana lutea L. var. aurantiaca. Las plantas de maíz y arroz se transformaron con los genes de los factores de transcripción ZmMYB y ZmPBF. Se analizó la expresión génica transitoria y se realizó transformación estable. Los resultados obtenidos indicaron que tanto ZmPBF como ZmGAMYB pueden transactivar la expresión de ZmBCH2 en endospermo de maíz, y ZmPBF y ZmGAMYB transactivar independientemente el promotor de ZmBCH2 en arroz. Los dos parálogos de IPPI (OsIPPI1 y OsIPPI2) aislados previamente en arroz tuvieron un patrón de expresión diferente; el ARNm de OsIPPI1 fue más abundante que el ARNm de OsIPPI2 en todos los tejidos. Se usó la microscopía de fluorescencia confocal y microscopía inmunoelectrónica para determinar la localización de ambas proteínas. Estas se localizan en el retículo endoplásmico (RE), así como en los peroxisomas y las mitocondrias, mientras que solo se detectó OsIPPI2 en los plastidios. La detección de ambas isoformas en el RE indica que DMAPP se puede sintetizar de novo en este compartimiento. Diferentes técnicas como UPLC, GC-MS y qRT-PCR también se utilizaron para perfilar los metabolitos primarios y secundarios y la expresión génica en plántulas de arroz des-etioladas. Los resultados revelaron que los genes involucrados en la en el metabolismo primario y secundario están regulados por la luz, especialmente en la biosíntesis de isoprenoides en hojas de arroz. Once derivados de pelargonidina se identificaron en los pétalos de G. lutea y se perfilaron los genes de la vía de biosíntesis, revelando que DFR, ANS y 3GT afectan principalmente a la acumulación de los glucósidos de pelargonidina. Todos estos resultados contribuyen al conocimiento, a diferentes niveles, de la regulación de las rutas biosinteticas de los carotenoides en plantas superiores.
This thesis mainly focuses on functional analysis and characterization of a number of secondary metabolite biosynthetic genes and the regulation of the corresponding secondary metabolite biosynthetic pathway in plants. The overall aims were to elucidate the transcriptional regulation of β-carotene hydroxylase 2 gene (BCH2) in maize, the functional analysis of rice isopentenyl diphosphate isomerases (OsIPPI), and determine their subcellular localization. Simultaneously, the influence of light on the metabolic pathway in rice was studied and the pelargonidin quantification and essential pelargonidin biosynthesis genes in Gentiana lutea L. var. aurantiaca were identified. Maize and rice plants were transformed with transcription factor genes ZmMYB and ZmPBF, via transient gene expression and stable transformation respectively. The results indicated that both ZmPBF and ZmGAMYB can transactivate ZmBCH2 expression in maize endosperm and ZmPBF and ZmGAMYB independently transactivate the ZmBCH2 promoter in rice. The two IPPI paralogs (OsIPPI1 and OsIPPI2) isolated previously in rice had a different expression pattern; OsIPPI1 mRNA was more abundant than OsIPPI2 mRNA in all tissues. Confocal fluorescence microscopy and immuno-electron microscopy were used to determine the localization of both proteins. These localized to the endoplasmic reticulum (ER) as well as peroxisomes and mitochondria, whereas only OsIPPI2 was detected in plastids. The detection of both isoforms in the ER indicates that DMAPP can be synthesized de novo in this compartment. UPLC, GC-MS and qRT-PCR were used to profile the primary and secondary metabolites and gene expression in de-etioleted rice seedlings. The results revealed both primary and secondary metabolism and the corresponding genes are regulated by light, especially isoprenoids biosynthesis in rice leaves. Eleven pelargonidin derivatives were identified in the petals of G. lutea and the biosynthetic pathway genes were profiled, revealing DFR, ANS and 3GT mainly affect the accumulation of pelargonidin glucosides. Collectively my results provide novel insights of the regulation of carotenoid and flavonoid biosynthesis in higher plants at different levels.

Dans notre laboratoire, nous avons récemment mis au point un système permettant de visualiser la prénylation des protéines in vivo, en utilisant une lignée cellulaire de tabac BY-2 transformée exprimant une protéine GFP inductible par la déxaméthasone et fusionnée en C-terminal avec le domaine basique d’une calmoduline de riz, portant un motif de géranylgéranylation (GFP-BD-CVIL) (Gerber et al. , 2009). L'inhibition de la voie du MEP et de la géranylgéranyl-transférase de type 1 (PGGT-1) par des inhibiteurs spécifiques se traduit par une délocalisation de la protéine GFP-BD-CVIL de la membrane plasmique au noyau. Par contre, l'inhibition de la voie du MVA n'affecte pas la localisation de la protéine. Des essais de complémentation chimique avec des intermédiaires biosynthétiques ont confirmé que les précurseurs du GGPP, pour l'isoprénylation de la protéine de fusion, sont fournis essentiellement par la voie du MEP. Afin d'optimiser ce système de visualisation, la lignée cellulaire exprimant la protéine GFP-BD-CVIL a été retransformée avec un vecteur inductible par l'estradiol, contrôlant l'expression d'une protéine mRFP qui marque le noyau des cellules. Cette nouvelle stratégie simplifie la quantification de cellules viables. Couplée à un système semi-automatique de comptage, elle réduit à la fois le temps d'analyse et le risque de biais générés par l'utilisateur. Finalement, le test a été adapté pour l'utilisation de plaques de verre à 96 puits, permettant le criblage d'inhibiteurs spécifiques de la voie du MEP, susceptibles d'avoir des applications en pharmacie et en agriculture, tels que des antibiotiques, des agents anti-paludisme ou des herbicides
Higher plants synthesize their isoprenoids through two different routes, the cytosolic mevalonic acid (MVA) pathway and the plastidial 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. By contrast, many human pathogenes rely exclusively on the MEP pathway for the synthesis of their isoprenoids. As this pathway does not occur in mammals, it represents an attractive target for the design of biopharmaceuticals and herbicides. Our group has recently established an in vivo visualization system for the geranylgeranylation of proteins, on the basis of a stably transformed tobacco BY-2 cell line, expressing a GFP fused to the prenylable, C-terminal basic domain of a rice calmodulin, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). The use of pathway-specific inhibitors revealed that inhibition of the MEP pathway, as well as inhibition of the geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. By contrast, the inhibition of the MVA pathway did not affect the localization. Complementation assays with pathway-specific intermediates further confirmed that the precursors for the prenylation of the fusion protein are predominantly provided by the MEP pathway. To optimize the initial test system, the existing cell line was transformed with an inducible vector, driving the expression of a nuclear marker protein that allows an easier quantification of total cells by image analysis software. Finally, the assay was adapted for the use of 96-well glass-bottom plates and provided us with an inexpensive, visual system to screen for potential inhibitors of the MEP pathway

Statins, drugs commonly used to lower serum cholesterol, have been shown to stimulate osteoblast differentiation and bone formation. By inhibiting HMG-CoA reductase (HMGCR) statins deplete the cellular isoprenoid biosynthetic pathway products farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Current thought in the field is that statins stimulate bone formation through the depletion of GGPP, since exogenous GGPP prevents the effects of statins on osteoblasts in vitro. We hypothesized that direct inhibition of GGPP synthase (GGPPS) would similarly stimulate osteoblast differentiation. Digeranyl bisphosphonate (DGBP), a specific inhibitor of GGPPS, decreased GGPP levels in MC3T3-E1 pre-osteoblasts and calvarial osteoblasts leading to impaired protein geranylgeranylation. In contrast to our hypothesis, DGBP inhibited the matrix mineralization of MC3T3-E1 cells and the expression of osteoblast differentiation markers in calvarial osteoblasts. The effect on mineralization was not prevented by exogenous GGPP. By inhibiting GGPPS, DGBP led to an accumulation of the GGPPS substrate FPP. We show that FPP and GGPP levels decreased during MC3T3-E1 and calvarial osteoblast differentiation, which correlated with decreased expression of HMGCR and FPP synthase. The decrease in FPP during differentiation was prevented by DGBP treatment. The accumulation of FPP following 24 h DGBP treatment correlated with activation of the glucocorticoid receptor, suggesting a potential mechanism by which DGBP-induced FPP accumulation may inhibit osteoblast differentiation. To further investigate whether FPP inhibits osteoblast differentiation, we utilized the squalene synthase (SQS) inhibitor zaragozic acid (ZGA), which causes a greater accumulation of FPP than can be achieved with GGPPS inhibition. ZGA treatment decreased osteoblast proliferation, gene expression, alkaline phosphatase (ALP) activity, and matrix mineralization of calvarial osteoblasts. Prevention of ZGA-induced FPP accumulation with HMGCR inhibition prevented the effects of ZGA on osteoblast differentiation. Treatment of osteoblasts with exogenous FPP similarly inhibited matrix mineralization. These results suggest that the accumulation of FPP negatively regulates osteoblast differentiation. While we did not find that specific depletion of GGPP stimulates osteoblast differentiation, we obtained evidence that GGPP does negatively regulate the differentiation of these cells. Exogenous GGPP treatment inhibited primary calvarial osteoblast gene expression and matrix mineralization. Interestingly, GGPP pre-treatment increased markers of insulin signaling, despite reduced phosphorylation of the insulin receptor (InsR). Inhibition of osteoblast differentiation by GGPP led to the induction of PPARã and enhanced adipogenesis in osteoblastic cultures, suggesting that GGPP may play a role in the osteoblast versus adipocyte fate decision. Adipogenic differentiation of primary bone marrow stromal cell (BMSC) cultures was prevented by DGBP treatment. Altogether these data present novel roles for the isoprenoids FPP and GGPP in the regulation of osteoblast differentiation and have intriguing implications for the isoprenoid biosynthetic pathway in the regulation of skeletal homeostasis.

Les isoprenoides bacteriens forment un ensemble de molecules variees derivant toutes du diphosphate d'isopentenyle. Dans une premiere partie, nous discutons les methodes d'isolations de triterpenoides bacteriens pentacycliques et proposons notre propre methode. Celle-ci appliquee au micro-organisme bradyrhizobium japonicum nous a permis de caracteriser trois nouveaux triterpenoides naturels deux methyltetrahymanols et un dimethyltetrahymanol. Nous avons montre que le donneur des methyles supplementaires est issu de la methionine. Dans un second temps nous avons etudie la voie de biosynthese des isoprenoides independante du mevalonate. Apres avoir rappele les acquis du laboratoire, nous affinons le schema biogenetique hypothetique en y incluant le methylerythritol. Nous avons en effet montre, par des experiences d'incorporation, que la biosynthese de ce tetrol est etroitement lie a celle du diphosphate d'isopentenyle. Le methylerythritol est donc un hemiterpene. Nous avons ajoute une autre enterobacterie a la liste des organismes possedant la voie de biosynthese isoprenique independante du mevalonate, montrant l'interet medical a long terme de ces travaux. Nous avons mis au point la synthese chimique d'esters phosphoriques d'isoprenoides et d'intermediaires hypothetiques de la voie de biosynthese independante du mevalonate dans le but d'effectuer des incorporations en systemes acellulaires.

Highly branched isoprenoid (HBI) alkenes are ubiquitous lipids which have been identified in numerous geochemical samples, ranging from recent sediments to ancient oils. At the outset of the current investigation, the diatomaceous algae Haslea ostrearia (C25 alkenes) and Rhizosolenia setigera (C25 or C30 alkenes) were the only reported biological sources of these compounds. However, there remained a poor correlation between isomers found in diatoms and those commonly reported in sediments and water column particles. In the present study, the structures of fourteen novel C25 HBI trienes, tetraenes and pentaenes, and four C30 HBI pentaenes and hexaenes have been rigorously characterised via gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy following isolation from diatoms. The GC-MS characteristics of the four novel C30 HBIs and eight of the novel C25 HBIs characterised herein show an excellent correlation with those of the HBIs commonly reported in sediments and water column particles. In contrast to the HBIs characterised previously, which all possess a saturated branch point and fixed double bond stereochemistry, the common isomers (C25 and C30) possess an unsaturated branch point and exhibit E/Z isomerism about a trisubstituted double bond. Three diatoms belonging to the Pleurosigma genus have been identified as HBI producers. Of these, the benthic species P. intermedium and the planktonic species Pleurosigma sp. have been found to biosynthesise the common C25 HBI isomers. P. planktonicum has also been identified as a producer of C25 HBIs possessing a novel structural type. The HBI distributions in five distinct strains of R. setigera have been investigated, and these were found to be highly variable. Two strains isolated from the northwest Atlantic were found to produce a single, uncommon C25 HBI pentaene, which has also been reported in H. ostrearia. In contrast, R. setigera isolated from the Arabian Sea was found to produce C30 HBIs only, whilst two strains isolated from southern Brittany were found to co-produce the common C25 and C30 HBI isomers. Four diatom species belonging to the Haslea genus have also been newly identified as producers of C25 HBI alkenes. The HBI distributions in H. salstonica, H. crucigera, H. pseudostrearia and Haslea sp. were examined, and HBI production appears to be widespread within the Haslea genus. All of the HBIs identified in these Haslea spp. were of the structural type previously observed in H. ostrearia, and thus do not correspond to the HBI isomers most commonly reported in sediments and particles. Hydrocarbon extracts isolated from sediments and particulates from the Arabian Sea, Cariaco Trench, Peru upwelling region and the Black Sea were examined by GC-MS, and the HBI isomers in these samples were identified

L'addition d'une chaine laterale phytyl aux chlorophylles, tocopherols, et a la phylloquinone est necessaire pour leur integration dans les membranes plastidiales. Nous avons clone un adnc codant pour une geranylgeranyl reductase qui a ete exprimee dans e. Coli. . Cette enzyme catalyse la reduction de la geranylgeranyl-chlorophylle-a en phytyl-chlorophylle-a et la reduction du geranylgeranyl pyrophosphate libre en phytyl pyrophosphate. Cette multifonctionnalite suggere que dans le plaste, la meme geranylgeranyl reductase est utilisee dans la voie de biosynthese des chlorophylles, tocopherols et phylloquinones. Nous avons clone puis exprime dans e. Coli deux adncs de poivron codant pour les enzymes catalysant la formation de la cryptoxanthine et de la zeaxanthine. Nous avons montre que ces deux enzymes fonctionnent grace au pouvoir reducteur de la ferredoxine. L'analyse de la sequence peptidique nous a permis de determiner les residus histidine susceptibles d'intervenir dans les liaisons de coordination avec le fer ou dans le transfert du pouvoir reducteur. La mutation de ces residus en alanine, abolit totalement l'activite hydroxylase. La biosynthese des carotenoides foliaires de nicotiana benthamiana a ete perturbee en faisant synthetiser par les feuilles de la capsanthine, une xanthophylle non native des chloroplastes, grace a l'utilisation d'un vecteur viral a arn. L'adnc codant pour la capsanthine-capsorubine synthase a ete place sous le controle transcriptionel d'un promoteur subgenomique d'un tobamovirus. Les feuilles de plantes transfectees presentent un phenotype orange ainsi qu'une tres forte accumulation de capsanthine. Ce phenomene est associe a une distorsion des empilements granaires. La capsanthine synthetisee dans les chloroplastes foliaires se retrouve exclusivement dans les complexes trimeriques et monomeriques des antennes collectrices d'energie du psii. Aucune difference n'est observee au niveau des centres reactionnels psi et psii. Ces resultats montrent que les antennes collectrices des plantes superieures sont capables d'incorporer fonctionnellement des carotenoides nouveaux.

At least fourteen C25 highly branched isoprenoid (HBI) alkenes have been previously reported in estuarine and coastal sediments from locations worldwide. The parent alkane structure has been proven but only a few of the double bond positions established previously. A wide body of evidence suggests that the compounds have a diatomaceous origin, with one report in the laboratory cultured diatom Haslea ostrearia. Alkenes with more than two double bonds appear to be rapidly removed from the hydrocarbon fraction of most sediments. There is evidence that some of the alkenes react rapidly with sulphur to form S-containing analogues. HBIs, both as hydrocarbons and S-containing analogues, may prove useful environmental indicators once the sources and exact structures have been established. A study of the temporal variations of HBI and carotenoid biomarker concentrations in Tamar sediments was conducted during 1994. The concentrations of C25 HBI alkenes exhibited strong negative correlations with those of /J-C2i:6 and fucoxanthin, common diatom markers. The strong inverse variance suggests that HBI concentrations are related to diatom populations in the Tamar estuary. Six C25 HBI alkenes ranging from a diene to hexaene have been isolated from ‘bulk' cultures of Haslea ostrearia and Caspian Sea sediments. The occurrence of HBIs in Haslea ostrearia is thus confirmed. Furthermore, the structures were unambiguously assigned by detailed nuclear magnetic resonance spectroscopy and mass spectral analysis, as well as by chemical degradation. The hydrocarbons are 2,6,10,14-tetramethyl-7-(3- methylpent-4-enyl)pentadec-5-ene; 2,10,l4-trimethyi-6-methylene-7-(3-methyIpent-4- enyl)pentadec-9-ene; 2,10,14-trimelhyl-6-methylene-7-(3-methylpent-4-enyl)pentadec- 9,13-ene; 2,10,14-trimethyl-6-methylene-7-(3-methylpent-4-enyl)pentadec-2,9,13-ene; 2,6,10,14-tetramethyl-7-(3-methyIpent-4-enyl)pentadec-2,5,9,13-ene and 2,6,10,14- tetramethyl-7-(3-methylenepent-4-enyl)pentadec-2,5,9,l3-ene. Structural elucidation of the HBI alkenes produced by Haslea ostrearia and comparison with sedimentary distributions suggests that the latter may arise from a facile double bond isomerisation of the biosynthetic compounds. The evidence presented suggests that inputs from Haslea ostrearia may account for at least eight of the dominant sedimentary HBI alkenes. The unusual behaviour exhibited by HBI alkenes during solid phase chromatography suggests that an incomplete record exists for HBI alkenes reported in the environment.

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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
Despite the evident importance of the Amazon forest in regulating the global climate, through processes like photosynthesis and evapotranspiration, little is known about gases synthesized in the secondary metabolism and emited by trees of this ecosystem that are involved in these processes, such as the volatile terpenoids (e.g. monoterpenes and sesquiterpenes). In the present study, from a method of collecting volatile terpenoids in situ, with a low collection time and collected air volume, combined with thermal desorption analysis with gas chromatography coupled to mass spectrometry (TD GC-MS), the composition of volatile terpenoids in tree trunks belonging to 15 species of Protium (Burm. f.) genus was determined. A library of volatile terpenoids was created and sixteen (16) monoterpenes were found, in addition to six (6) sesquiterpenes. The monoterpenes α-pinene and D-limonene were the most abundant, appearing 63 and 58 times, respectively, in 69 samples. In addition, a new record of a monoterpene for tropical tree species, the ξ-fencheno, has been reported. An explanatory analysis of the distribution of volatile terpenoids by species was also performed, from which information on the richness of compounds in the genus Protium was obtained, as well as how each species could be characterized by the higher or lower frequency of compounds reported. Results of the present study provide a basis for the beginning of filling the knowledge gap on this subject, considering that to date no work on the characterization of volatile terpenoids in tropical tree species in the Brazilian Amazon has been found. In addition, these outcomes favor the understanding of the ecological and evolutionary interactions of resins, to ensure the sustainable commercial exploration of this material, as well as contribute to its ecological conservation. In addition, they may contribute to elucidate the functions of these compounds in the tropical forests dynamics (plant-plant interactions, plant-insect interactions) as well as the forest-atmosphere interactions.
Apesar da notória importância da Floresta Amazônica na regulação do clima do planeta, por meio de processos como a fotossíntese e evapotranspiração, pouco se conhece a respeito dos gases sintetizados e emitidos pelas árvores desse ecossistema, a partir do metabolismo secundário, envolvidos nesses processos, como é o caso dos terpenóides voláteis (e.g. monoterpenos e sesquiterpenos). No presente estudo, a partir de um método de coleta de terpenóides voláteis in situ, com baixo tempo de coleta e volume de ar coletado, aliado à análise por dessorção térmica com cromatografia a gás acoplada a espectrometria de massas (TD GC- MS, na sigla em inglês), foi possível determinar a composição de terpenóides voláteis em troncos de árvores resinosas pertencentes a 15 espécies do gênero Protium (Burm. f.). Uma biblioteca de terpenóides voláteis foi criada, sendo que 16 monoterpenos foram encontrados, além de 6 sesquiterpenos. Os monoterpenos α-pineno e D-limoneno foram os mais abundantes, sendo relatados 63 e 58 vezes, respectivamente, num total de 69 amostras. Ademais, um novo registro de monoterpeno para espécie arbórea tropical, o ξ-fencheno, foi relatado. Uma análise explanatória da distribuição de terpenóides voláteis por espécie também foi realizada, a partir da qual obteve-se informações sobre a riqueza de compostos no gênero Protium, além de como cada espécie pôde ser caracterizada pela maior ou menor frequência de compostos relatada. Os resultados do presente estudo fornecem uma base para o início do preenchimento de uma lacuna de conhecimento acerca dessa temática, tendo em vista que, até o momento não foram encontrados trabalhos sobre a caracterização de terpenóides voláteis em espécies arbóreas tropicais na Amazônia brasileira. Além disso, favorecem o entendimento sobre as interações ecológicas e evolutivas das resinas, visando assegurar a exploração comercial sustentável desse material, bem como contribuir para sua conservação ecológica. Ainda, podem contribuir para a obtenção de maiores esclarecimentos sobre as funções desses compostos na dinâmica das florestas tropicais (interações planta-planta; interações planta-inseto), bem como nas interações floresta-atmosfera.

Os isoprenóides são compostos essenciais a todos os organismos e representam um grupo extremamente amplo estruturalmente e funcionalmente. Em plantas, mais de 30.000 compostos desta classe foram identificados até hoje. Todas as plantas produzem isoprenóides que desempenham papéis essenciais como os carotenóides, as clorofilas e as plastoquinonas (fotossíntese); ubiquinona (respiração); citocininas, brassinosteróides, giberelinas, ácido abscísico (regulação do crescimento e desenvolvimento). No entanto, a maior variedade de isoprenóides é representada pelos metabólitos secundários (óleos essenciais como eucaliptol, cineol, citronelal). Os isoprenóides possuem papel marcante nas relações da planta com o ambiente onde estão inseridas, já que medeiam as relações planta-inseto, planta-microorganismo e planta-planta. Devido ao valor comercial dos compostos isoprenóides, há grande interesse em produzi-los por bioengenharia em bactérias ou em plantas e o entendimento do papel dos genes e proteínas codificadas na rota de síntese dessas unidades básicas de formação de terpenóides é extremamente importante. Nesse trabalho, nós recuperamos, a partir da seleção dos plasmídios de clonagem pSPORT1 (Invitrogen) purificados do Projeto “Genolyptus: Rede Brasileira de Pesquisa do Genoma de Eucalyptus”, os clones selecionados e potencialmente codificadores de 1-desoxi-D-xilulose-5-fosfato redutoisomerase (DXR) e de outras enzimas atuantes nas rotas de síntese de isoprenóides, como 1-desoxi-D-xilulose 5-fosfato sintase (DXS), mevalonato difosfato descarboxilase (MDC), isopentenil difosfato isomerase 1 (IPPI1) e isopentenil difosfato isomerase 2 (IPPI2). Nos estoques de plasmídios do projeto Genolyptus, foi possível encontrar as sequências completas dos genes dxr, ippi1 e ippi2. Os genes foram analisados in silico e a sequência de ippi1 foi utilizada na modelagem molecular e dinâmica molecular para avaliação de características peculiares do folding protéico desse tipo de proteína eucariótica. Os genes foram clonados em vetores pGEX-4T (GE Healthcare) e heterologamente expressos em Escherichia coli. Foi realizada, também, uma análise transcricional comparativa dos genes selecionados pela técnica de microarranjos.
Isoprenoids are essential to all organisms and are the most structurally and functionally diverse group of plant metabolites. In plants, more than 30,000 compounds of this class were identified to date. All plants produce isoprenoids that can play essential roles as carotenoids, chlorophylls and plastoquinone (photosynthesis); ubiquinone (respiration); regulation of growth and development (cytokinins, brassinosteroids, gibberellins, abscisic acid). However, the majority of isoprenoids is represented by secondary metabolites (essential oils like eucaliptol, cineol, citronelal). Isoprenoids have important roles in the relationships between plants and the environment, since they can mediate plant-insect, plant-microorganism and plant-plant interactions, as well as participate in abiotic stress responses. Due to the high value of isoprenoid compounds, there is great interest in producing them by bioengineering in bacteria or plants. Understanding the role of genes and proteins related to the biosynthetic pathway of isoprenoids is extremely important. In this work, we retrieved, from the collection of cloning plasmids pSPORT1 (Invitrogen) generated in the “Genolyptus Project: The Brazilian Research Network on the Eucalytpus Genome”, selected clones that potentially codified the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and other enzymes from the isoprenoid biosynthetic pathway including 1-deoxy-D-xylulose 5-phosphate synthase (DXS), mevalonate disphosphate decarboxilase (MDC), isopentenil disphosphate isomerase 1 (IPPI1) and isopentenil disphosphate isomerase 2 (IPPI2). Complete sequences of the dxr, ippi1 and ippi2 genes were succesfully recovered from the Genolyptus stocks. The genes were analyzed in silico and the ippi1 sequence was used in studies of molecular modeling and molecular dynamics in order to evaluate specific folding characteristics of this kind of eukaryotic IPPI. The genes were cloned into pGEX-4T vectors (GE Healthcare) and expressed in Escherichia coli. Also, the transcriptional analysis of the selected genes was performed by microarray analysis.

In addition to the production of phytol (from chlorophylls) and sterols, a limited number of diatom species are capable of synthesising unusual C25 and C30 highly branched isoprenoid (HBl) alkenes. At the outset of the current investigation, the structures of most C25 and C30 HBIs had been identified. Some environmental factors had been shown to control their production, although a detailed understanding of these remained unclear. In addition, the biological functions of the chemicals remained unknown, and the reasons for their production by some species and not by others, was not understood. Investigations on the distributions of C25 and C30 HBl alkenes biosynthesised by Rhizosolenia setigera demonstrated a dependence on the physiological status of the cells, as measured by the position of this diatom in its life cycle. Thus, while C30 HBIs were observed at every stage of the life cycle, C25 HBIs were not always present in the cells. Since the synthesis of C25 HBIs appears to be stimulated by the onset of auxosporulation (sexual reproduction), an explanation is provided as to why they have rarely been observed in previous studies. Two novel monocyclic C30 alkenes (previously reported in other strains of Rhizosolenia setigerd), and a novel monocyclic C25 alkene were also observed during life cycle experiments. The two C30 hydrocarbons structures were subsequently characterised and the potential geochemical relevance of these compounds was highlighted by comparison of their mass spectral and chromatographic properties with those of alkenes reported in sediments and suspended water column particles. An investigation of terpenoid (including HBl) biosynthesis in the diatoms Haslea ostrearia, Rhizosolenia setigera and Pleurosigma intermeclium has been performed. Evidence for species and organelle dependent biosynthetic pathways has been observed. Phytol is synlhesised by each species investigated according to the recently discovered methyl-erythritol phosphate (MEP) pathway. This pathway is also involved in the synthesis of C25 HBIs in the two species Haslea ostrearia and Pleio-osigma intermedium. In contrast, C25 and C30 HBIs, and (at least) one monocyclic C30 alkene, appear to be made predominantly via the mevalonate (MVA) route in the diatom R. setigera. Evidence for the contribution of the MVA pathway to the biosynthesis of sterols was found for the diatoms Rhizosolenia setigera, and Pleurosigma intermedium. In contrast, only contributions from the MEP pathway were found for the biosynthesis of sterols in Haslea ostrearia. Preliminary evidence for dynamic interchange between the two pathways has also been observed. Fractionation of Rhizosolenia setigera cells revealed that phytol was present in the chloroplasts, while sterols and HBIs were present in the cytoplasm.

The nitrogenous bisphosphonates pamidronate, alendronate, risedronate, and zoledronate are used clinically in the treatment of bone disease. All of these drugs inhibit the enzyme farnesyl diphosphate synthase (FDPS), which mediates production of farnesyl diphosphate (FPP). However, because it is a branch point in isoprenoid biosynthesis, FPP is involved in the biosynthesis of several different substrates at the same time. One key enzyme downstream of FDPS in isoprenoid biosynthesis is geranylgeranyl diphosphate synthase (GGDPS) which affords the geranylgeranyl diphosphate (GGPP) necessary for prenylation of the small GTPases such as Ras, Rab, Rho and Rac, that are important signaling proteins. Non-nitrogenous analogues of the clinical drugs, including mono- and bisisoprenoid bisphosphonates, have been developed more recently. These new analogues have been found to inhibit GGDPS selectively. Because it is important to inhibit the generation of GGPP, selective inhibition of GGDPS is highly desirable. In previous research, digeranyl bisphosphonate (DGBP) was discovered to show good inhibition of GGDPS. In order to obtain more potent analogues of the compound DGBP, and to study the biological effect of an á-alkoxy group on bisphosphonate compounds, a series of ether bisphosphonates has been prepared and studied. A second important enzyme in isoprenoid biosynthesis is geranylgeranyl transferase II (GGTase II). This enzyme transfers GGPP to Rab proteins, and thus converts the parent proteins to lipoproteins which are essential for their proper cellular localization. One known inhibitor of this enzyme is the chemical 3-PEHPC, but a high concentration of this compound is necessary to generate any cellular effects. In an effort to study the cellular effects that result from inhibition of this enzyme, and to develop more potent inhibitors, my research has focused on modification of 3-PEHPC to obtain derivatives that may have improved biological activity. Both the known compounds 3-PEHPC and 3-PEPC, and new structures, including the first generation PEHPC N-oxides and the second-generation compounds prepared through click chemistry, have been prepared and tested for activity in this system.

Thesis (Ph. D.) -- University of Maryland, College Park, 2006.
Thesis research directed by: Cell Biology & Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Plant sesquiterpene synthases catalyze the conversion of the linear substrate farnesyl diphosphate, FPP, into a remarkable array of secondary metabolites. These secondary metabolites in turn mediate a number of important interactions between plants and their environment, such as plant-plant, plant-insect and plant-pathogen interactions. Given the relative biological importance of sesquiterpenes and their use in numerous practical applications, the current thesis was directed towards developing a better understanding of the mechanisms employed by sesquiterpene synthases in the biosynthesis of such a diverse class of compounds. Substrate preference for sesquiterpene synthases initially isolated from Nicotiana tabacum (TEAS), Hyoscyamus muticus (HPS) and Artemisia annuna (ADS) were optimized with regards to a divalent metal ion requirement. Surprisingly, careful titration with manganese stimulated bona fide synthase activity with the native 15-carbon substrate farnesyl diphopshate (FPP) as well as with the 10-carbon substrate geranyl diphosphate (GPP). Reaction product analysis suggested that the GPP could be used to investigate early steps in the catalytic cascade of these enzymes. To investigate how structural features of the sesquiterpene synthases translate into enzymatic traits, a series of substrate and active site residue contacts maps were developed and used in a comparative approach to identify residues that might direct product specificity. The role and contribution of several of these residues to catalysis and product specificity were subsequently tested by the creation of site-directed mutants. One series of mutants was demonstrated to change the reaction product to a novel sesquiterpene, 4-epi-eremophilene, and while another series successfully transmutated TEAS into a HPS-like enzyme. This is the first report of a rational redesign of product specificity for any terpene synthase. The contact map provides a basis for the prediction of specific configurations of amino acids that might be necessary for as yet uncharacterized sesquiterpene synthases from natural sources. This prediction was tested by the subsequent isolation and validation that valencene synthase, a synthase from citrus, did indeed have the amino acid configuration as predicted. Lastly, an in vitro system was developed for analyzing the interaction between sesquiterpene synthases and the corresponding terpene hydroxylase. Development of this in vitro system is presented as a new important tool in further defining those biochemical features giving rise to the biological diversity of sesquiterpenes.

Los isoprenoides son una de las familias más grande de metabolitos en la naturaleza y son especialmente diversos en el reino vegetal. Entre ellos, los carotenoides, los tocoferoles y la filoquinona tienen un interés especial por su papel como vitaminas. Las plantas sintetizan estos compuestos en cloroplastos para contribuir a la fotosíntesis y fotoprotección. En el caso de los carotenoides, sus niveles más altos se encuentran en plastos especializados llamados cromoplastos, que se encuentran típicamente en órganos pigmentados no verdes como pétalos de flores y frutos maduros, pero solo ocasionalmente en hojas. El objetivo general de esta tesis ha sido probar nuevas estrategias para el enriquecimiento de verduras en vitaminas isoprenoides a través de un sistema desarrollado en nuestro laboratorio para inducir la conversión de cloroplastos en cromoplastos en hojas. La herramienta se basa en la gran capacidad de la enzima crtB de la bacteria Pantoea ananatis para producir fitoeno y la capacidad de las hojas para convertir este fitoeno adicional en carotenoides posteriores con cambios concomitantes en la ultraestructura de los plástos. La disponibilidad de este sistema permitió definir dos objetivos específicos: (1) caracterizar el contexto fisiológico del fenotipo inducido por crtB en hojas de Nicotiana benthamiana y (2) probar diferentes estrategias aprovechando este sistema basado en crtB para mejorar la biofortificación de hojas. En la primera parte de la tesis, demostramos que el fenotipo no reversible de diferenciación de cloroplasto a cromoplasto desencadenado por crtB en plastos se asocia con una rápida pérdida de actividad fotosintética causada por la acumulación de fitoeno. Este fenómeno hace que el cloroplasto pase a ser competente para la cromoplastogénesis. Luego, la transición se completa una vez que el fitoeno se convierte en carotenoides posteriores mediante enzimas endógenas. También demostramos que los tratamientos que causan un equilibrio redox alterado de los fotosistemas y un estrés oxidativo facilitan la diferenciación de los cromoplastos. En la segunda parte de la tesis, caracterizamos los cambios estructurales asociados con la diferenciación de cromoplastos mediada por crtB en hojas. Durante el proceso, los plastoglóbulos aumentan en número y tamaño y se utilizan para almacenar fitoeno y otros isoprenoides, incluidos β-caroteno (pro-vitamina A), tocoferoles (vitamina E) y filoquinona (de vitamina K). Los plastoglobulos son el sitio de localización y acción de la proteína crtB y demostramos que las condiciones que estimulan la proliferación de plastoglóbulos (como la luz intensa) se pueden utilizar para promover aún más la acumulación de vitaminas isoprenoides. Nuestros resultados muestran que la combinación de crtB con genes involucrados en la biosíntesis de dichas vitaminas puede aumentar aún más sus niveles. Por último, mostramos que β-caroteno se puede acumular aún más combinando la cromoplastogénesis mediada por crtB con una vía extraplastidial sintetica. También demostramos que las optimizaciones del sistema crtB se pueden aplicar para biofortificar vegetales de verduras como la lechuga, contribuyendo así al desarrollo de nuevos alimentos.
Els isoprenoides són una de les famílies més gran de metabòlits en la naturalesa i són especialment diversos en el regne vegetal. Entre ells, els carotenoides, els tocoferols i la filoquinona tenen un interès especial pel seu paper com vitamines. Les plantes sintetitzen aquests compostos en cloroplasts per contribuir a la fotosíntesi i fotoprotecció. En el cas dels carotenoides, els seus nivells més alts es troben en plastidis especialitzats anomenats cromoplastos, que es troben típicament en òrgans pigmentats no verds com pètals de flors i fruits madurs, però només ocasionalment en fulles. L’objectiu general d’aquesta tesi ha estat provar noves estratègies per a l’enriquiment de verdures en vitamines isoprenoides a través d’un sistema desenvolupat en el nostre laboratori per induir la conversió de cloroplasts en cromoplastos en fulls. L’eina es basa en la gran capacitat de l’enzim crtB del bacteri Pantoea ananatis per produir fitoe i la capacitat de les fulles per convertir aquest fitoe addicional en carotenoides posteriors amb canvis concomitants a la ultraestructura dels plastidis. La disponibilitat d’aquest sistema va permetre definir dos objectius específics: (1) caracteritzar el context fisiològic de l’fenotip induït per crtB en fulls de Nicotiana benthamiana i (2) provar diferents estratègies aprofitant aquest sistema basat en crtB per millorar la biofortificación de fulles. A la primera part de la tesi, vam demostrar que el fenotip no reversible de diferenciació de cloroplast a cromoplast desencadenat per crtB en plastidis s’associa amb una ràpida pèrdua d’activitat fotosintètica causada per l’acumulació de fitoe. Aquest fenomen fa que el cloroplast passi a ser competent per a la cromoplastogénesis. Després, la transició es completa una vegada que el fitoe es converteix en carotenoides posteriors mitjançant enzims endògenes. També vam demostrar que els tractaments que causen un equilibri redox alterat dels fotosistemes i un estrès oxidatiu faciliten la diferenciació dels cromoplastos. A la segona part de la tesi, caracteritzem els canvis estructurals associats amb la diferenciació de cromoplastos intervinguda per crtB en fulls. Durant el procés, els plastoglóbuls augmenten en nombre i mida i s’utilitzen per emmagatzemar fitoe i altres isoprenoides, inclosos β-carotè (provitamina A), tocoferols (vitamina E) i filoquinona (de vitamina K). Els plastoglobuls són el lloc de localització i acció de la proteïna crtB i vam demostrar que les condicions que estimulen la proliferació de plastoglóbuls (com la llum intensa) es poden utilitzar per promoure encara més l’acumulació de vitamines isoprenoides. Els nostres resultats mostren que la combinació de crtB amb gens involucrats en la biosíntesi d’aquestes vitamines pot augmentar encara més els seus nivells. Finalment, vam mostrar que β-carotè es pot acumular encara més combinant la cromoplastogénesis intervinguda per crtB amb una via extraplastidial sintètica. També vam demostrar que les optimitzacions de sistema crtB es poden aplicar per biofortificar vegetals de verdures com l’enciam, contribuint així a el desenvolupament de nous aliments.
Isoprenoids are one of the largest families of metabolites in nature, and they are especially diverse in the plant kingdom. Among them, carotenoids, tocopherols and phylloquinone are interesting for their role as vitamins. Plants synthesize these compounds in chloroplasts to contribute to photosynthesis and photoprotection. In the case of carotenoids, their highest levels are found in specialized plastids named chromoplasts, which are typically found in non-green pigmented organs such as flower petals and ripe fruits but only occasionally in leaves. The general goal of this thesis has been testing new strategies for the enrichment of leafy vegetables in isoprenoid vitamins through a system developed in our lab to induce the conversion of chloroplasts into chromoplasts in leaves. The tool is based on the capacity of the crtB enzyme from the bacterium Pantoea ananatis to boost the production of phytoene and the ability of leaves to convert this extra phytoene into downstream carotenoids with the concomitant changes in plastid ultrastructure. The availability of this system allowed to define two specific objectives: (1) to characterize the physiological context of the crtB-induced phenotype in Nicotiana benthamiana leaves and (2) to test different strategies exploiting this crtB-based system to improve leaf biofortification. In the first part of the thesis, we demonstrate that the non-reversible chloroplast-to-chromoplast differentiation phenotype triggered by plastid-localized crtB is associated with a rapid loss of photosynthetic activity caused by the accumulation of phytoene. This phenomenon makes the chloroplast competent for chromoplastogenesis. The transition is then completed once phytoene is converted into downstream carotenoids by endogenous enzymes. We also demonstrate that treatments that cause altered redox balance of the photosystems and oxidative stress facilitate chromoplast differentiation. In the second part of the thesis, we characterize the structural changes associated with crtB-mediated chromoplast differentiation in leaves. During the process plastoglobules increase in number and size. Plastoglobules are used to store phytoene and other isoprenoids (including pro-vitamin A β-carotene, vitamin E tocopherols, and vitamin K phylloquinone. We also show that plastoglobules are the site of localization and action of the crtB protein and demonstrate that conditions that promote plastoglobule proliferation (such as high light) can be used to further promote the accumulation of isoprenoid vitamins. Our results show that the combination of crtB with genes involved in the biosynthesis of such vitamins can further increase their levels. Lastly, we show that β-carotene can be further accumulated by combining the crtB-mediated chromoplastogenesis with an engineered extraplastidial pathway. We also show that the optimizations of the crtB system can be applied to biofortify edible green leafy vegetables such as lettuce, hence contributing to the development of new functional foods.
Universitat Autònoma de Barcelona. Programa de Doctorat en Biologia i Biotecnologia Vegetal

Thesis (Ph. D.)--University of Kentucky, 2003.
Title from document title page (viewed onJune 1, 2004). Document formatted into pages; contains x, 145 p. : ill. Includes abstract and vita. Includes bibliographical references (p. 126-143).

Environmental issues related to the use of fossil resources point to the need to obtain energy from sun, wind and biomasses. Among these sources, biomasses have many advantages, including the ability to be storable and ubiquitous. Agro-processing industry generates a large amounts of residues that can be exploited to obtain bioactive compounds and fine chemicals. In this context, the supply chain strategy should evolve towards the biorefinery concept, an integrated manufacturing platform from which to obtain, from a specific crop, different types of bioproducts. This PhD work is aimed to the development of expeditious and scalable methods to isolate bioactive compounds (mainly isoprenoids) from agro-industrial biomasses and to set the basis for their use as fine chemicals in various realms of application (pharma, food, cosmetics). Chemistry and bioactivity of two types of isoprenoids have been investigated, namely sesquiterpenoids of the germacrone and bisabolone clan, and triterpenoids from the pentacyclic-(ursane, oleanane, lupane, glycirrhetinic) and the tetracyclic (cycloartane) type. The sources are by-products from agriculture (guayule resin, olive tree prunings) or horticultural (plane bark) activities, or food plants (matè, licorice, turmeric). When see under an antropocentric light, my thesis has been an attempt to “domesticate” these compounds, tailoring them to specific biomedical indications in the realm of chronic diseases (inflammation, metabolic disease, cancer).

A malária mata mais de um milhão de pessoas por ano, sendo uma das doenças infecciosas mais relevantes e um grande problema de saúde pública. Além disso, o surgimento de cepas resistentes aos quimioterápicos utilizados faz necessário o estudo de novos alvos para tratamentos contra esta doença. No nosso laboratório foi demonstrada a biossíntese de isoprenóides, em P. falciparum pela via MEP. Sabe-se que substâncias inibidoras da biossíntese de isoprenóides, dentre essas os terpenos, apresentam atividade antimalárica. Levando em consideração o anterior, nós avaliamos o potencial antimalárico do álcool perilico (POH) em P. falciparum e P. berghei. Nossos resultados demonstraram que o POH teve efeito inibitório contra o crescimento do P. falciparum in vitro, nas cepas 3D7 e K1 com uma IC50 de 4,8 ± 0,5 μM, e 10,41±2,33 μM, respectivamente. Além disso, o POH não teve efeito tóxico na linhagem celular Vero. Ainda, Comprovamos que o POH inibiu a farnesilação de proteinas entre 20 e 37 KDa de P. falciparum. Por outro lado, os experimentos in vivo não mostraram eficácia do tratamento do POH contra PbGFP em camundongos Balb/c. Em contraste, foi demostrada a eficácia do POH na de malária cerebral experimental (MCE), , indicando uma redução na taxa de incidência da MCE no grupo tratado com POH, comparado o não tratado ( P<0,05). Além disso, o POH reduziu a inflamação no cérebro dos animais tratados, uma vez que teve uma redução significativa na adesão de leucócitos aos vasos cerebrais (P<0.001), como também, o numero de hemorragias foi menor comparados com os animais não tratados. (P<0.0001). Portanto, os resultados obtidos nesta pesquisa abrem novas alternativas no estudo do mecanismo de ação do POH como um terpeno com grande potencial para tratar MC.
Malaria kills over one million people a year worldwide, and is one of the most important infectious diseases and a major public health problem. Furthermore, the emergence of resistant strains to chemotherapeutic agents used, make it necessary to study new targets for treatments against this disease. In our laboratory we have demonstrated the isoprenoids biosynthesis in P. falciparum, by the MEP pathway. It is known that the substances that inhibit isoprenoid biosynthesis, among these terpenes, have antimalarial activity in vitro and in vivo. Considering this, we evaluate the antimalarial potential of PA (POH) in P. falciparum and P. berghei. Our results showed that the POH had inhibitory effect against the growth of strains 3D7 and K1 of P. falciparum in vitro, with an IC50 of 4.8 μM ± 0.5, and 10.41 ± 2.33 μM, respectively. Furthermore, the POH had no toxic effect on cell line Vero. Moreover, the POH proved that inhibited proteins farnesylation from 20 to 37 kDa of P.falciparum. On the other hand, in vivo experiments did not show efficacy on treatment against POH PbGFP in BALB/c mice. In contrast, the effectiveness of POH in the experimental cerebral malaria (MCE) was demonstrated, indicating a reduction in the incidence rate of MCE in the group treated with POH, compared with of untreated animals (P <0.05). In addition, the POH reduced inflammation in the brain of treated animals, since it had a significant reduction in leukocyte adhesion to cerebral vessels (P <0.001), as also the number of bleeding was lower compared to untreated animals (P<0.0001). Therefore, the results obtained in this work provide new alternatives to study the POH\'s mechanism of action as a terpene with great potential to treat MC.

The condensation of stable 2-(1-(trimethylsilyl)ethylidene)malononitrile with elemental sulfur was explored in the parallel-synthesis of a small library of structurallydiverse2-aminothiophenesandthieno[2,3-d]pyrimidines. Bromination and ipso-iododesilylation of these heterocyclic scaffolds provided synthetic methods to efficiently generate key intermediates, allowing for great versatility. Bisphosphonic acid derivatives of thieno[2,3-d]pyrimidine and benzothiazole scaffolds, with favourable physicochemical properties, were evaluated for their ability to inhibit isoprenoid biosynthesis and potentially modulate the function of small G-proteins implicated in a range of human diseases. Preliminary biological activities and selectivity will be presented.
La condensation de 2-(1-(trimethylsilyl)ethylidene)malononitrile avec du soufre élémentaire a été explorée dans le parallèle synthèse d'une petite group de structures diverses de 2-aminothiophenes et thieno[2,3-d]pyrimidines. Bromation et ipso-iododesilylation de ces échafaudages hétérocyclique fourni des méthodes de synthèse pour générer efficacement des intermédiaires clés, ce qui permet une grande polyvalence. Dérivés d'acide bisphosphonique de thieno[2,3-d]pyrimidines et échafaudages benzothiazole, avec de bonnes propriétés physico-chimiques, ont été évalués pour leur capacité à inhiber la biosynthèse des isoprénoïdes et potentiellement moduler la fonction des petites protéines G impliquées dans un assortiment de maladies humaines. Les activités préliminaires biologiques et la sélectivité sera présenté.