Dissertations / Theses on the topic 'Nucleotide sugars'

UDP-sugar metabolizing pyrophosphorylases provide the primary mechanism for de novo synthesis of UDP-sugars, which can then be used for myriads of glycosyltranferase reactions, producing cell wall carbohydrates, sucrose, glycoproteins and glycolipids, as well as many other glycosylated compounds. The pyrophosphorylases can be divided into three families: UDP-Glc pyrophosphorylase (UGPase), UDP-sugar pyrophosphorylase (USPase) and UDP-N-acety lglucosamine pyrophosphorylase (UAGPase), which can be discriminated both by differences in accepted substrate range and amino acid sequences. This thesis focuses both on experimental examination (and re-examination) of some enzymatic/ biochemical properties of selected members of the UGPases and USPases and UAGPase families and on the design and implementation of a strategy to study in vivo roles of these pyrophosphorylases using specific inhibitors. In the first part, substrate specificities of members of the Arabidopsis UGPase, USPase and UAGPase families were comprehensively surveyed and kinetically analyzed, with barley UGPase also further studied with regard to itspH dependency, regulation by oligomerization, etc. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Frc-1-P, whereas USPase reacted with arange of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P, β-L-Ara-1-P and α-D-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P and, to some extent, with D-Glc-1-P. A structure activity relationship was established to connect enzyme activity, the examined sugar-1-phosphates and the three pyrophosphorylases. The UGPase/USPase/UAGPase active sites were subsequently compared in an attempt to identify amino acids which may contribute to the experimentally determined differences in substrate specificities. The second part of the thesis deals with identification and characterization of inhibitors of the pyrophosphorylases and with studies on in vivo effects of those inhibitors in Arabidopsis-based systems. A novel luminescence-based high-throughput assay system was designed, which allowed for quantitative measurement of UGPase and USPase activities, down to a pmol per min level. The assay was then used to screen a chemical library (which contained 17,500 potential inhibitors) to identify several compounds affecting UGPase and USPase. Hit-optimization on one of the compounds revealed even stronger inhibitors of UGPase and USPase which also strongly inhibited Arabidopsis pollen germination, by disturbing UDP-sugar metabolism. The inhibitors may represent useful tools to study in vivo roles of the pyrophosphorylases, as a complement to previous genetics-based studies. The thesis also includes two review papers on mechanisms of synthesis of NDP-sugars. The first review covered the characterization of USPase from both prokaryotic and eukaryotic organisms, whereas the second review was a comprehensive survey of NDP-sugar producing enzymes (not only UDP-sugar producing and not only pyrophosphorylases). All these enzymes were discussed with respect to their substrate specificities and structural features (if known) and their proposed in vivo functions.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2010.
Includes bibliography.
ENGLISH ABSTRACT: The study of plants on a sub-cellular level is an important, yet challenging area and its application allows for novel insight into the understanding of metabolism and its regulation. In this study I describe the development of a reverse phase liquid chromatography mass spectrometry (RPLC-MS) technique in which 29 phosphorylated and nucleotide sugars could be detected and quantified. The method was validated with the use of authentic standards and the system displayed very good linearity (Rª > 0.95), while the recovery of the standards added to the plant material before extraction was between 65 and 125%. Further, Arabidopsis thaliana wild type (Col-0) and adenylate kinase (adk1) mutant leaf discs were fed 13C labeled glucose over a period of 24 hours and harvested at defined time intervals. Non aqueous fractionation, and metabolite profiling via the above mentioned rpLC-MS method in conjunction with gas chromatography mass spectrometry (GC-MS) allowed for the detection and quantification of primary metabolites on a sub-cellular level as well as the determination of their relative isotopic label enrichments through primary carbon metabolism. Finally, a yeast complementation system was designed for the identification of tonoplast bound sucrose import proteins. The screening system identified 22 unique sequences from an Arabidopsis thaliana cDNA library. Four unknown sequences were identified, one of which displayed tonoplast membrane association upon in silico analysis. Three ATP-binding proteins were also identified as well as a sub-unit from the exocyst gene family. Further studies will include the functional characterization of the latter, as well as the development of additional cDNA libraries more suited for screening of sequences that encode sucrose importer proteins.
AFRIKAANSE OPSOMMING: Die studie van plante op a sub-sellulere vlak is ‘n belangrike maar uitdagende navorsingsarea en die toepassing daarvan dra by tot unieke insig tot ‘n beter begrip van metabolise regulasie. In die studie bespreek ek die ontwikkeling van ‘n teenoorgestelde fase vloeistof kromatografie massa spektrometrie (RPLC-MS) tegniek waarin 29 gefosforileerde en nukleotied suikers gevind en gekwantifiseer kon word. Geldigverklaring van die metode is bewerkstelling met die gebruik van oorspronklike standaarde and die systeem het baie goeie liniariteit (Rª > 0.95) getoon, terwyl die herstelbaarheid van standaarde wat bygevoeg is by die plant material voor ekstraksie tussen 65% en 125% was. Arabidopsis thaliana wilde type (Col-O) en die adenaliet kinase (adk1) mutant blaar dele is met 13C gemerkte glukose gevoed oor ‘n tydperk van 24 uur en geoes by spesifieke tydstippe. Nie-vloeibare fraksionering en metaboliet uitleg is vermag vanaf die genoemde RPLC-MS metode met behulp van gas kromotografie massa spektrometrie (GC-MS) wat die bepaling en kwantifikasie van primere metaboliete op n sub-sellulere vlak sowel as die bepaling van hul relatiewe isotropiese merker verrykers deur primere metabolisme toelaat. Verder is n gis komplementere systeem ontwerp vir die identifikasie van tonoplas gebinde sukrose invoer proteine. Die verkenningsysteem het 22 unieke volgordes opgelewer vanaf ‘n Arabidopsis thaliana kDNA biblioteek. Vier onbekende volgordes is geidentifiseer, een wat tonoplas membraan assosiasie toon met in silico analise. Drie ATP-bindings proteine is ook geidentifiseer asook ‘n sub-eenheid van die eksosyst geen familie. Verdere studies sal die funksionele karakterisering van die laaste protein insluit, asook die ontwikkeling van additionele kDNA biblioteke meer gepas vir verkenning sodiende identifiseer van volgordes wat sukrose invoer proteine vertaal.

A parede celular de plantas é formada basicamente por celulose, hemicelulose e lignina. A formação dos polímeros de hemicelulose depende do suprimento de precursores chamados de açúcares-nucleotídeos. A biossíntese das diferentes estruturas de hemicelulose da parede celular envolve a participação de enzimas pertencentes às famílias das glicosiltransferases (GTs). Estudos feitos em Arabidopsis thaliana, Brachypodium distachyon, Oryza sativa (arroz) e Zea mays (milho) auxiliaram na descoberta de 11 enzimas da via de interconversão nucleotídeo-açúcar e de enzimas da família das glicosiltransferases (GTs), como as GT2, GT8, GT43, GT47, GT61 e GT75, envolvidas na biossíntese de hemicelulose. O presente trabalho visa a identificação de genes da via de biossíntese de hemicelulose da parede celular de cana-de-açúcar (Saccharum spp.) e análise filogenética entre Arabidopsis thaliana (planta modelo de eudicotiledôneas), Oryza sativa, Brachypodium distachyon, Zea mays, Sorghum bicolor e Saccharum spp. Foram identificados os genes das famílias GT2, GT8, GT43, GT47, GT61, GT75, CSL, Epimerase e UDPG em cana-de-açúcar a partir da busca em sete bibliotecas de RNA-Seq utilizando as sequências de O. sativa, Z. mays e S. bicolor como referência. Os domínios específicos de cada família gênica foram confirmados através do programa PFAM e consequentemente anotados. A identificação e anotação das sequencias possibilitou a construção de bancos de sequências das famílias envolvidas na biossíntese de hemicelulose para as espécies A. thaliana, B. distachyon, O. sativa, Z. mays e S. bicolor. Foram identificadas para cada espécie, respectivamente, um total de 67, 49, 49, 60 e 56 genes bona fides. O presente trabalho, além da identificação de genes nas diferentes espécies, permitiu a identificação e seleção de 27 genes candidatos envolvidos na biossíntese de hemicelulose em cana-de-açúcar e possivelmente envolvidos na recalcitrância da parede celular nas diferentes bibliotecas de RNA-Seq de cana-de-açúcar.
The plant cell wall is mainly composed of cellulose, hemicellulose and lignin. The formation of hemicellulose polymers lies on the supply of the so-called sugar-nucleotide precursors. The diverse hemicellulose structures biosynthesis of cell wall involves the participation of enzymes belonging to the families of glycosyltransferases (GTs). Studies in Arabidopsis thaliana, Brachypodium distachyon, Oryza sativa (rice) and Zea mays (corn) aid the discovery of 11 enzymes of the nucleotide sugar interconversion pathway and enzymes of the GT family, as GT2, GT8, GT43, GT47, GT61 e GT75, involved in the hemicelluloses biosynthesis. This study aims to the identification of hemicellulose biosynthesis pathway genes from the cell wall of sugarcane (Saccharum spp.) and phylogenetic analysis of Arabidopsis thaliana (eudicotyledonous plant model), Oryza sativa, Brachypodium distachyon, Zea mays, Sorghum bicolor and Saccharum spp. The genes of the GT2, GT8, GT 43, GT47, GT61, GT75, CSL, epimerase and UDPG families were identified in sugarcane from a search in seven RNA-Seq libraries using the sequences of O. sativa, Z. mays and S. bicolor as reference. The specific domains of each gene family have been confirmed through the PFAM program and consequently noted. The identification and annotation of the sequences enabled the construction of sequences banks of the families involved in hemicellulose biosynthesis in the species A. thaliana, B. distachyon, O. sativa, Z. mays and S. bicolor. It was identified for each species, respectively, a total of 67, 49, 49, 60 and 56 bona fides genes. This work, in addition to the identification of genes in different species, allowed the identification and selection of 27 candidate genes involved in the biosynthesis of hemicelluloses in sugarcane and possibly involved in cell wall recalcitrance in the different sugarcane RNA-Seq libraries.

Myxococcus xanthus displays social (S) motility, a form of surface motility that is key to the multicellular behaviors of this organism. S motility requires two cellular structures: type IV pili (TFP) and exopolysaccharides (EPS). Previous studies have shown that M. xanthus does not use glucose or any other sugar as a primary carbon source. However, eight monosaccharides, namely glucose, mannose, arabinose, galactose, xylose, rhamnose, N-acetyl-glucosamine, and N-acetyl-mannosamine, are found in M. xanthus EPS. In this study, pathways that M. xanthus could use to produce the activated sugar monomers to form EPS are proposed based on genomic data. Of the eight sugars, pathways for seven were disrupted by mutation and their effects on the EPS-dependent behaviors were analyzed. The results indicate that disruption of the two pathways leading to the production of activated rhamnose (GDP- and TDP-rhamnose) affected fruiting body formation (GDP form only) and dye binding ability (both forms) but not S motility. Disruptions of the xylose, mannose, and glucose pathways caused M. xanthus to lose S motility, fruiting body formation, and dye binding abilities. An interruption in the pathway for galactose production created a mutant with properties similar to a lipopolysaccharide (LPS) deficient strain. This discovery led us to study the phenotypes of all mutant strains for LPS production. The results suggest that all mutants may synthesize defective LPS configurations. Disruption of the UDP-N-acetyl-mannosamine pathway resulted in a wild type phenotype.

In addition, it was discovered that interruption of the pathway for N-acetyl-glucosamine production was possible only by supplementing this amino-sugar in the growth medium. In an attempt to determine if other mutants could be recovered by sugar supplementation, it was discovered that the Î pgi mutant can be rescued by glucose supplementation. The Dif chemotaxis-like pathway is known to regulate EPS production in M. xanthus. DifA is the upstream sensor of the pathway. Previous studies had created a NarX-DifA chimeric protein, NafA, that enables the activation of the Dif pathway by nitrate, the signal for NarX. In this study, we constructed a Î pgi difA double mutant containing NafA. This strain was then subjected to various incubations with glucose and/or nitrate to determine whether the point of EPS regulation by the Dif pathway is down- or up-stream of the step catalyzed by Pgi (phosphoglucose isomerase). Preliminary results from this study are inconclusive.

Master of Science

The project aim was to determine which of the competing pathways predominate(s) in vivo in the formation of the UDP-GlcA, GDP-GalA and UDP-Man utilised for plant cell wall synthesis. The studies conducted on the ³H:¹⁴C ratios of plant cell wall residues at 8 h indicate that UDP-Gal is not a significant direct precursor of UDP-GalA. The ³H:¹⁴C ratios of the intermediary metabolites were studied over time. The results show that the ³H:¹⁴C ratio kinetics of UDP-GalA and UDP-Gal are vastly different from one another. These results also indicate that UDP-Gal is not a significant direct precursor of UDP-GalA. The ³H:¹⁴C ratios of the AIR (alcohol insoluble residue)-derived monosaccharides indicate that UDP-GlcA, UDP-GalA, UDP-Xyl, UDP-Ara and UDP-Api, like UDP-Rha, arose mainly from the UDP-Glc ‘core’ metabolite. The ³H:¹⁴C ratio kinetics of UDP-GalA, UDP-GlcA, UDP-Xyl and UDP-Ara are very distinct from the ³H:¹⁴C ratio kinetics of Glc 6-P but they are similar to the isotope ratio kinetics of UDP-Glc. The ³H:¹⁴C ratios of the AIR-derived monosaccharides and ³H:¹⁴C ratios of the intermediary metabolites give strong evidence that UDP-GalA and UDP-GlcA are predominantly formed by the UDP-Glc dehydrogenase pathway and not the myo-inositol pathway. The ³H:¹⁴C ratios kinetics of UDP-Glc are approximately equal to those of Glc 1-P. It is observed that the ³H:¹⁴C ratio of AIR-derived GalA residue is greater than for Man and Fuc. As UDP-GalA was determined to arise predominantly from UDP-Glc, GDP-Man and GDP-Fuc cannot also stem from Glc 1-P. The predominant pathway of GDP-Man synthesis must be via Gla 6-P or Fru 6-P. The ³H:¹⁴C ratio of Rib was similar to those of GalA, Ara, Xyl and Api. A pathway is known to exist that may convert to UDP-Xyl to the RNA precursor Rib 5-P. The results suggest that this is the predominant pathway of Rib synthesis for RNA.

The analysis of gene changes associated with formation of the mycorrhizal symbiosis between orchid and fungi could have broad implications for plant pathogen interactions. Fungi associated with North American terrestrial orchids were once included in the pathogenic genus Rhizoctonia. This suggests that orchids are able to overcome or utilize normally pathogenic pathways to establish symbioses. A differential display technique was employed to analyze gene changes in orchid in response to a fungus. Samples of RNA from roots of Cypripedium parviflorum var. pubescens (CyPP) grown in the presence or absence of a mycorrhizal fungus; Thanatephorus pennatus, were analyzed using AFLP differential display. Forty-four fragments were selected out of 5000 as being differentially expressed, but only 15 sequences were obtained. Most showed homology to ribosomal genes. Two represented genes believed to be regulated by the mycorrhizal interaction: trehalose-6-phosphate synthase/phosphatase (Tps), which showed down-regulation and nucleotide binding protein (NuBP), which showed up-regulation. The Tps partial clone identifies 2100 bp at the 3' end of the gene and encodes a protein of 667 amino acids. The NuBP gene is approximately 1200bp in length and encodes a protein of 352 amino acids. The Tps gene exists in multiple copies with high expression in roots and low expression in rhizomes and leaves. The NuBP gene exists as a single copy and has a low level of expression in rhizomes and leaves. Expression of Tps is induced by sucrose, but reduced by trehalose. Cultivation of CyPP with non-mycorrhizal fungi did not affect expression of Tps or NuBP. Trehalose induced NuBP expression whereas sucrose did not. A second species of mycorrhizal fungi induced expression of NuBP but reduced expression of Tps. Analysis of Tps expression in Arabidopsis was done using promoter:GUS fusions. The Tps promoter:GUS plants revealed that Tps expression is constitutive in roots. Regulation of Tps driven GUS is expressed throughout seedlings. GUS was not detected in leaves of older plants but was detected in anthers and stigmatic surfaces of flowers. Expression of GUS driven by Tps showed a strong wound response and was present in the junction between siliques and pedicels.
Ph. D.

Els glicolípids són producte d’alt valor degut a les seves propietats amfipàtiques que els doten d’un ampli rang d’aplicacions en els sectors químic (ex., biosurfactants) o biomèdic (ex., adjuvant de vacunes). Depenent de la unitat lipídica que els forma aquests compostos poden ser classificats en diferents famílies. Si la unitat lpídica és una ceramida o diacilglicerol, el glicolípid resultant es coneixerà com a glicoesfingolípid o glicoglicerolípid (GGL) respectivament. Mentre que els glicoesfingolípids han demostrat jugar un paper clau en diversos processos biològics, els glicoglicerolípids són interessants degut al seu ús potencial per a ser usats com a adjuvants de vacunes o supressors tumorals. Tot i que l’interès per aquests compostos és alt, la seva aplicació es veu obstaculitzada per la seva baixa disponibilitat i alt cost de producció. La síntesi química requereix de complexes passos de protecció i desprotecció per tal d’aconseguir la desitjada regio- i estereoespecificitat de l’enllaç glicosídic que, conseqüentment, comporta una reducció del rendiment i eficiència del procés. Per això, vam considerar l’enginyeria metabòlica com a estratègia potencial per a la producció de glicolípids i ens vam centrar en l’obtenció d’una plataforma d’enginyeria metabòlica en E. coli per tal d’obtenir aquests complexes productes d’interès. En estudis previs, el nostre grup va reportar que la sintasa de glicolípids MG517 de Mycoplasma genitalium era funcional i que s’obtenien glicoglicerolípids a partir de UDP-glucosa (UDP-Glc) i diacilglicerol (DAG). Addicionalment, una primera generació de soques modificades va demostrar que la disponibilitat de DAG era limitant per a la producció de GGL (Mora-Buyé et al., 2012). En el present projecte, cinc estratègies diferents d’enginyeria metabòlica van ser proposades per tal d’augmentar la producció de GGL utilitzant E. coli. Les primeres quatre estratègies tenien com a objectiu incrementar el pool del precursor lipídic, DAG. Sent així, la primera estratègia es basà en augmentar la disponibilitat de DAG a través de l’eliminació de reaccions competitives. Per aconseguir-ho, es van knockejar diferents gens involucrats en la ß-oxidació i l’activació d’àcids grassos (∆tesA i ∆fadE) reportant un increment en la producció de casi el doble. La segona estratègia es va basar en incrementar la disponibilitat d’àcids grassos mitjançant la modulació de factors de transcripció (fabR i fadR). Aquesta estratègia no va reportar un increment de la producció però si un canvi en el perfil lipídic amb un increment d’àcids grassos insaturats. La tercera estratègia es basava en incrementar la conversió dels donadors d’acils a àcid fosfatídic, precursor del DAG, sobreexpressant les aciltransferases PlsC i PlsB. La quarta estratègia es centrà en augmentar la disponibilitat del diacilglicerol per la sobreexpressió de la proteïna de fusió PlsCxPgpB, capaç de redirigir el flux cap a DAG, o CDH promovent la hidròlisi de fosfolípids. D’entre les diferent soques modificades, ∆tesA co-expressant MG517 i la proteïna de fusió PlsCxPgpB va ser la soca més productora, amb un 350% d’increment en la producció de GGL comparant-la amb la soca parental expressant únicament MG517. Especialment interessant és que les soques coexpressant CDH van presentar un canvi en el perfil de GGL cap al lípid diglucosilat (representant al voltant d’un 80% del total de GGLs). Finalment, es va proposar una estratègia metabòlica per incrementar la disponibilitat de l’altre precursor, UDP-Glc. Aquesta cinquena estratègia es va basar en sobre expressar l’enzim GalU, responsable de la biosíntesi d’UDP-Glc, i eliminant l’enzim codificant per la UDP-sucre difosfatasa ushA. No obstant, cap d’aquestes modificacions va aconseguir millorar els nivells de GGLs. Per últim, tal i com va ser reportat pel nostre grup que la fosfatidiletanolamina era intercanviable en les membranes d’E. coli pels nous producte GGL, una llibreria de promotors i RBS va ser dissenyada per tal de disminuir la producció d’aquest fosfolípid, augmentant al mateix temps de la producció de glicolípids.
Los glicolípidos son productos de alto valor debido a sus propiedades amfipáticas, que los dota en un amplio rango de aplicaciones en los sectores químicos (ej., biosurfactantes) o biomédicos (ej., adyuvante de vacunas). Dependiendo de la unidad lipídica que los forma, los glicolípidos son clasificados en diferentes familias. Si la unidad lipídica es una ceramida o diacilglicerol, los glicolípidos son conocidos como glicoesfingolípidos o glicoglicerolípidos (GGL) respectivamente. Mientras que los glicoesfingolípidos han demostrado jugar papeles clave en diversos procesos biológicos, los glicoglicerolípidos son compuestos interesantes debido a su potencial uso como adyuvantes de vacunas o supresores tumorales. Aunque el interés por estos compuestos es muy alto, su aplicación se ve obstaculizada por su baja disponibilidad y altos costes de producción. La síntesis química requiere de complejos pasos de protección y desprotección para conseguir la deseada regio- y estereoespecificidad del enlace glicosídico, que conlleva a una reducción del rendimiento y eficiencia del proceso. Por ello, consideramos la ingeniería metabólica como estrategia potencial para la producción de glicolípidos y nos centramos en construir una plataforma de ingeniería metabólica en E. coli para conseguir estas complejas estructuras de interés. En previos estudios, nuestro grupo reportó que la sintasa de glicolípidos MG517 de Mycoplasma genitalium era funcional y que glicoglicerolípidos podían ser obtenidos a partir de UDP-glucosa (UDP-Glc) y diacilglicerol (DAG). Adicionalmente, la primera generación de cepas modificadas demostró que la disponibilidad de DAG era limitante en la producción de GGL (Mora-Buyé et al., 2012). En el presente proyecto, cinco estrategias diferentes de ingeniería metabólica fueron propuestas para aumentar la producción de GGL en E. Coli. Las primeras cuatro estrategias se centraron en aumentar el pool del precursor lipídico, DAG. Para ello, la primera estrategia se basó en incrementar la disponibilidad de DAG a través de la eliminación de reacciones competitivas. Para lograrlo, se knockearon diferentes genes relacionados con la ß-oxidación y la activación de ácidos grasos (∆tesA y ∆fadE) reportando un incremento de casi el doble. La segunda estrategia se basó en incrementar la disponibilidad de ácidos grasos mediante la modulación de factores de transcripción (fabR y fadR). Aunque estrategia no reportó una mejora en el rendimiento de GGL, sí mostró un cambio en el perfil de los ácidos grasos con un incremento de los ácidos grasos insaturados. La tercera estrategia se basó en incrementar la conversión de los donadores de acilos a ácido fosfatídico, precursor del DAG, mediante la sobreexpresión de las aciltransferasas PlsC y PlsB. La cuarta estrategia se centró en aumentar la disponibilidad de diacilglicerol mediante la sobreexpresión de la proteína de fusión PlsCxPgpB capaz de redirigir el flujo de DAG, o CDH promoviendo la hidrólisis de fosfolípidos. Entre las diferentes cepas modificadas, la cepa ∆tesA coexpresando MG517 y la proteína de fusión PlsCxPgpB fue la mayor productora, con un incremento de los niveles de GGL del 350%, comparándola con la cepa parental expresando únicamente MG517. Interesantemente, las cepas coexpresando CDH mostraron un cambio en el perfil de GGL hacia el lípido diglucosilado (hasta el 80% del total del GGLs). Finalmente, una estrategia metabólica fue propuesta para aumentar la disponibilidad del otro precursor, UDP-Glc. La quinta estrategia se basó en la sobreexpresión de la enzima GalU, responsable de la biosíntesis de UDP-Glc, y la eliminación de la UDP-azúcar difosfatasa codificada por el gen ushA. Sin embargo, ninguna de estas modificaciones mejoró los niveles de GGL. Por último, tal y como reportó nuestro grupo que la fosfatidiletanolamina era intercambiable en las membranas de E. coli por los nuevos compuestos GGL, una librería de promotores y RBS fue diseñada para disminuir la producción de este fosfolípido intentando al mismo tiempo aumentar la producción de glicolípidos.
Glycolipids are products of high-added value due to their amphipathic properties, which endow them with a broad range of applications in the chemical (i.e., biosurfactants) and biomedical sectors (i.e., vaccine adjuvants). Depending on their lipidic moiety, glycolipids are classified in different families. If the lipid moiety is a ceramide or diacylglycerol, the glycolipids are known as glycosphingolipids or glycoglycerolipids respectively. While glycosphingolipids have shown to play essential roles in many biological processes, glycoglycerolipids (GGL) are interesting compounds due to their potential use as vaccine adjuvants or tumor suppressors. Although the interest of these compounds is very high, their applications are hampered by their low availability and high productions costs. Chemical synthesis requires complex protection and deprotection steps to achieve the desired regio- and stereospecificity of the glycosidic linkage, which consequently lower the yield and efficiency of the process. Therefore, we considered metabolic engineering as a potential strategy for the production of glycolipids and we aimed at building up a metabolic engineering platform in E. coli to achieve these complex structures of interest. In previous studies, our group reported that the glycolipid synthase MG517 from Mycoplasma genitalium was functional and glycoglycerolipids were obtained from UDP-glucose (UDP-Glc) and diacylglycerol (DAG). In addition, the first generation of engineered strains demonstrated that the availability of the DAG was the key bottleneck in GGL production (Mora-Buyé et al., 2012). In the present project, five different metabolic strategies were proposed to increase the production of GGL using E. coli. The first four strategies were aimed at increasing the available pool of the lipidic precursor, DAG. Thus, the first strategy was based on increasing DAG availability by removing competing reactions. To achieve so, different genes involved in the ß-oxidation and activation of fatty acids were knocked out (ΔtesA y ΔfadE) reporting an almost 2-fold production increase. The second strategy was based on increasing fatty acids availability by modulating different transcriptional factors (fabR y fadR). Although this strategy did not report an improvement of GGL yield, showed a change in the fatty acid profile with an increase of unsaturated fatty acids. The third strategy was based on increasing the conversion of acyl donors to phosphatidic acid, precursor of DAG, by overexpressing PlsC and PlsB acyltransferases. The fourth strategy was based on increasing diacylglycerol availability by overexpressing the fusion PlsCxPgpB protein that could redirect the flux to DAG or CDH promoting the hydrolysis of phospholipids. Among the different engineered strains, the ∆tesA strain co-expressing MG517 and a fusion PlsCxPgpB protein was the best producer, with a 350% increase of GGL titer compared to the parental strain expressing MG517 alone. Interestingly, the strains co-expressing CDH showed a shift in the GGL profile towards the diglucosylated lipid (up to 80% of total GGLs). Finally, a metabolic strategy was proposed to increase the availability of the other precursor, UDP-Glc. This fifth strategy was based on overexpressing GalU enzyme, which is responsible for the biosynthesis of UDP-Glc, and by removing the UDP-sugar diphosphatase encoding gene ushA. However, none of these modifications further improved the GGL titers. Finally, as it was also reported by our group that phosphatidylethanolamine was exchangeable in the membranes of E. coli by the new GGL compounds, a library of promoters and RBS was designed to decrease the production of this phospholipid trying at the same time to increase the production of glycolipids.

A parede celular vegetal, estrutura essencial para as plantas, é extremamente importante para a economia humana, já que apresenta diversas utilidades, como por exemplo, fabricação de papel, fibras de vestuário, construção civil, entre outras. A maior parte da parede celular vegetal primária (aproximadamente 90%), é formada por polissacarídeos como celulose, hemiceluloses e pectinas. Os monossacarídeos, unidades formadoras dos polissacarídeos, são sintetizados, nas plantas, a partir de diferentes açúcares nucleotídeos, sendo que, o suprimento desses, pode afetar a biossíntese dos polissacarídeos da parede celular. Visando analisar o impacto da alteração do fluxo metabólico do carbono na composição da parede celular, o presente projeto de pesquisa teve como objetivo alterar a composição dos polissacarídeos da parede celular de Nicotiana tabcum, através da modulação da expressão do gene uxs, responsável pela codificação da enzima UDP-D-glucuronato descarboxilase (UDPGlcADC, EC 4.1.1.35) que converte UDP-D-glucuronato em UDP-D-xilose, importante açúcar nucleotídeo, precursor do monossacarídeo xilose. Para isso, após a clonagem do gene uxs de ervilha, foram obtidas plantas transgênicas de tabaco superexpressando esse gene. Diversas análises foram realizadas para determinação da composição química da parede celular primária e secundária dessas plantas. Pela análise de FTIR da parede celular primária, verificou-se que três linhagens transgênicas apresentaram espectrotipos consistentes, indicando uma redução na quantidade de pectinas e ligações ésteres carboxílica nessas linhagens transgênicas. Apesar de não terem sido detectadas alterações na proporção dos monossacarídeos ramnose, xilose, arabinose, manose e galactose, e na quantidade de celulose, na parede celular primária das plantas transgênicas, foram observadas diferenças na proporção de galactose não esterificada, nas linhagens que apresentaram espectrotipo. Com relação à parede celular secundária, observou-se que algumas linhagens transgênicas apresentaram maior concentração de lignina solúvel relacionada a uma redução no conteúdo de lignina insolúvel.
The plant cell wall is not only an essential structure for plants, but also an extremely important raw material in human economy. The plant cell wall has diverse utilities, for example, papermaking, textile fiber, civil construction. Polysaccharides, such as cellulose, hemicelluloses and pectins, are the major components of the primary plant cell wall (approximately 90%). These polysaccharides are formed by monosaccharides, which are synthesized in the plant from different nucleotide sugars. The suppliment of the nucleotide sugars can affect plant cell wall polysaccharides biosynthesis. Aiming at analyzing the impact of the alteration in the metabolic carbon flux on cell wall composition, the objective of this research project was to alterate the plant cell wall polysaccharides composition by the modulation of the uxs gene. This gene encodes the UDP-D-glucuronic acid decarboxylase enzyme (UDPGlcADC, EC 4.1.1.35) that promotes the conversion of UDP-D-glucuronic acid to UDP-D-xylose, an important sugar nucleotide precursor of xylose monosaccharide. To achieve this goal, the pea uxs gene was cloned and transgenic tobacco plants overexpressing this gene were obtained. Several analyses were performed to determinate the primary and secondary cell wall composition of those transgenic plants. The primary cell wall analysis by FTIR identified three transgenic lines that show different spectrotypes compared to wild type and those transgenic spectrotypes had the same features. The results indicate a reduction of pectin and ester carbonyl binding in the transgenic plants. No alterations were detected in the monosaccharide (rhamnose, xylose, arabinose, manose and galactose) proportions and the amount of cellulose in the primary cell wall of the transgenic plants. Nevertheless, differences in the proportion of unesterified galactose were observed in the same transgenic lines that showed spectrotypes. With regard to secondary cell wall, some transgenic lines showed an increase in soluble lignin which is related to a reduction in insoluble lignin.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
In this study, the effects of Aluminium (Al) and of different diets, both individually and in association, were investigated through the determination of NTPDase, 5 -nucleotidase and acetylcholinesterase (AChE) activities in rat brain. In addition, we investigated said effects on oxidative stress by determining activity of the antioxidant enzyme catalase as well as lipid peroxidation by measuring TBARS levels. Male rats were exposed to Al (50 mg/kg/day) by gavage during three months. NTPDase and 5 -nucleotidase activities were then determined in synaptosomes of cerebral cortex and hippocampus, as well as in platelets. ATP, ADP and AMP hydrolysis was increased in both synaptosomes of cerebral cortex and hippocampus, as well as in platelets. AChE activity and TBARS levels were determined in homogenate of different brain structures in mice exposed to Al (2.7 mg/kg/day) by gavage during three months. The group that received Al+sodium citrate presented an increase in AChE activity in hippocampus, striatum, cortex and hypothalamus. On the other hand, the group that received only Al presented a decrease in AChE activity in hypothalamus and an increase in striatum. Moreover, AChE was determined in S1 of different brain structures, synaptosomes of cerebral cortex and erythrocytes of male rats exposed to Al (50 mg/kg/day) by gavage during three months. There was an increase in AChE activity in S1 of striatum and hypothalamus and in synaptosomes of cerebral cortex and erythrocytes. However, in S1 of cerebellum, hippocampus and cortex there was a decrease. In addition, the effect of diets rich in saturated fat and refined sugar on AChE activity in homogenate of different brain structures, on catalase activity in liver and on TBARS levels in plasma and liver were determined in female and male rats. There was a decrease in AChE activity in hippocampus, cortex and hypothalamus of male and female rats given both a diet rich in saturated fat and a diet rich in refined sugar. There was no alteration of AChE activity in cerebellum and striatum. For both diets, catalase activity was increased in liver of male and female rats. In addition, considering the alterations brought about by the individual exposure to both environmental factors, Al and diets, the effect of the association of both factors was evaluated. Thus, after a period of three months of exposure to both Al (50 mg/kg/day) by gavage and diets rich in saturated fat and saturated/polyunsaturated fat ad libitum, NTPDase and 5 -nucleotidase activities were determined in synaptosomes of cerebral cortex and platelets of rats. Animals receiving both diets in association with Al and Al/Ci presented an increase in ATP, ADP and AMP hydrolysis in synaptosomes of cerebral cortex and platelets. The results obtained in the present study demonstrate that exposure to both environmental factors, Al and diets rich in saturated fat and refined sugar, either individually or in association, affected the purinergic and cholinergic systems and caused oxidative stress in rats.
Neste estudo, investigamos os efeitos do alumínio (Al) e de diferentes dietas, individualmente e em associação, através da determinação da atividade das enzimas NTPDase, 5 -nucleotidase e acetilcolinesterase (AChE) no encéfalo de roedores. Adicionalmente, investigamos o estresse oxidativo, através da atividade da enzima antioxidante catalase, e a peroxidação lipídica pela medida dos níveis de TBARS. Ratos machos foram expostos ao Al (50 mg/kg/dia) através de gavagem, por um período de 3 meses. Após o tratamento, foi determinada a atividade das enzimas NTPDase e 5 -nucleotidase em sinaptossoma de córtex cerebral, hipocampo e plaquetas. A hidrólise dos nucleotídeos ATP, ADP e AMP foi aumentada, nas frações sinaptossomais de córtex cerebral e hipocampo bem como nas plaquetas. A atividade da AChE e os níveis de TBARS foram determinados em homogeneizado de diferentes estruturas cerebrais de camundongos expostos ao Al (2,7 mg/kg/dia), através de gavagem, por um período de 3 meses. Quanto à atividade da AChE, o grupo que recebeu Al+citrato de sódio apresentou um aumento da atividade desta enzima em hipocampo, estriado, córtex e hipotálamo. Já o grupo que recebeu só Al apresentou uma diminuição da atividade em hipotálamo e um aumento em estriado. Além disto, a atividade da AChE foi determinada em S1 de diferentes estruturas cerebrais, sinaptossoma de córtex cerebral, e em eritrócitos de ratos machos expostos à Al (50 mg/kg/dia), através de gavagem, por 3 meses. A atividade da AChE apresentou um aumento em S1 de estriado e hipotálamo, e em sinaptossoma de córtex cerebral e eritrócitos. Porém, em sobrenadante (S1) de cerebelo, hipocampo e córtex houve uma diminuição. Adicionalmente, foi determinado o efeito de dietas ricas em gordura saturada e açúcar refinado sobre a atividade da enzima AChE em homogeneizado de diferentes estruturas encefálicas, e atividade da enzima catalase em fígado, e os níveis de TBARS em plasma e fígado de ratos machos e fêmeas. A atividade da AChE em hipocampo, córtex e hipotálamo de ratos machos e fêmeas foi diminuída, após exposição a ambas as dietas, rica em gordura e rica em açúcar. Nas estruturas cerebelo e estriado não houve alteração na atividade da AChE. Após o consumo de ambas as dietas, a atividade da enzima catalase foi aumentada em fígado de ratos machos e fêmeas. Adicionalmente, considerando todas as alterações ocasionadas pela exposição individual aos fatores ambientais, Al e dietas, foi avaliado o efeito da associação entre esses dois fatores. Dessa forma, após um período de 3 meses de exposição conjunta ao Al (50 mg/kg/dia) através de gavagem e o consumo ad libitum de dietas ricas em gordura saturada e gordura saturada/poliinsaturada, foram determinadas a atividade das enzimas NTPDase e 5 - nucleotidase em sinaptossomas de córtex cerebral e plaquetas de ratos. Os animais que receberam ambas as dietas administradas em conjunto com Al e Al/Ci apresentaram um aumento na hidrólise dos nucleotídeos ATP, ADP e AMP, em sinaptossoma de córtex cerebral e plaquetas. Os resultados obtidos no presente estudo relatam que a exposição de roedores a ambos os fatores ambientais Al e dietas ricas em gordura saturada e açúcar refinado, individualmente e em conjunto, afetam os sistemas purinérico e colinérgico, e causam estresse oxidativo.

Sugar nucleotides are essential building blocks for the glycobiology of all organisms being intrinsically linked to cellular metabolism. However, the market price of sugar nucleotides is currently very high due to the inexistence of cost-effective manufacturing processes. In this thesis, an in vitro enzyme process for the synthesis of sugar nucleotides UDP-Man, UDP- Glc and UDP-GlcNAc using low cost substrates was developed and studied in order to decrease the manufacturing costs of sugar nucleotides. The effects of pH and buffer composition on the cascades were investigated leading to an increase in the productivity of the target product. Another importante discovery was the enzymatic production of the sugar nucleotide, UDP-Man, representing the first step towards the more economical production of this sugar. Moreover, it was found that, by adjusting the concentration of the enzymes in the cascades, the productivity of the UDP-Glc and the UDP-GlcNAc cascade could be increased by 52% and 56%, respectively. Finally, it was observed that when high concentration of product if formed, in situ removal of sugar nucleotide might be necessary. Given the results obtained, this thesis presents new data and ideas towards the establishment of a cost-effective in vitro enzymatic production process of UDP-Man, UDP-Glc and UDP-GlcNAc with very promising practical applications.

The study of many of glycosyltransferases is limited due to an inadequate access to sugar nucleotides. Preparation of sugar nucleotides through the use of nucleotidylyltransferases with broad substrate specificities is gaining significant interest and offers high yields and stereospecificity. Physiologically, the glucose 1-phosphate thymidylyltransferase catalyzes the condensation of ?-D-glucose 1-phosphate and deoxythymidine triphosphate to yield deoxythymidine diphospho glucose. Exploiting and targeting these enzymes also has the potential of yielding new therapeutics. Cps2L is a thymidylyltransferase isolated from Streptococcus pneumoniae, with broad substrate flexibility. The substrate specificity of Cps2L was evaluated with new sugar 1-phosphate analogues to gain further insight into substrate and inhibitor requirements. Several sugar 1-phosphate analogues including sugar 1C-phosphonates (and analogues thereof), 2-deoxy-2-fluorosugar 1-phosphates, and glucopyranose 1- boranophosphates have been used to probe the sugar 1-phosphate modification tolerance of Cps2L. In addition, NMR spectroscopy was used to determine the anomeric stereochemistry of 2-deoxy-2-fluorosugars nucleotide products. For those substrates that were accepted by Cps2L, steady-state kinetic parameters were determined. The enzyme is able to almost equally form Michaelis complexes with different sugar substrates, whereas the turnover values for obtaining the corresponding sugar nucleotide were different. The evaluation of the substrate tolerance of Cps2L, as well as the synthesis of ?-D-glucose-1C-thiophosphonate, a difluoro and a bisphosphono analogue of ?-D-glucose 1C-phosphonate will be described.

博士
國立清華大學
化學系
100
Abstract Carbohydrates and their glycoconjugates are important in mediating structural and functional roles in numerous physiological processes, including various disease states. Despite significant advancement in the field like programmable one-pot assembly of carbohydrates, at the recent time, synthesis of complex carbohydrates and glycoconjugates remains elusive than that of other biomolecules. To simplify the synthesis of carbohydrates, enzymes provide an alternative means that are likely to be synthetically viable to chemists. In this regard, enzymes like glycosyltransferases and glycosidases have proven useful biocatalysts in constructing stereo- and regiospecific glycosidic linkages in complex carbohydrate structures. However, in the preparative-scale synthesis point, glycosyltransferases from microbial sources may exhibit greater flexibility because of their ability to synthesize a large range of oligosaccharide analogues at relatively high yields. My dissertation describes the expression of various recombinant bacterial enzymes; thymidylyltransferase (RmlA) of Aneurinibacillus thermoaerophilus, N-acetylhexosamine-1-kinase (NahK) of Bifidobacterium longum, β-1,3-N-acetyl-glucosaminyltransferase of Helicobacter pylori (HpGnT) and β-1,4-galactosyltransferase of Neisseria meningitides (NmGalT), from Escherichia coli. We determined that use of magnesium (Mg2+) as a cofactor and at 55 oC, numerous sugar nucleotides were effectively synthesized in milligram-scale by RmlA in two hours, and these include uridine 5′-diphosphate galactose (UDP-Gal), uridine 5′-diphosphate N-acetylglucosamine (UDP-GlcNAc), uridine 5′-diphosphate glucose (UDP-Glc) and thymidine 5′-diphosphate glucose (TDP-glucose). Additionally, RmlA was site-specifically and covalently immobilized on an MNP using a combination of intein-mediated protein expression and NCL, and found that Rm1A-MNP retains almost 95% of its activity following ten consecutive enzyme assays. We also demonstrated synthesis of UDP-GlcNAc and UDP-Gal by using corresponding kinases from relatively cheap starting materials such as GlcNAc and Gal. All sugar nucleotides were purified by ion-exchange column for analytical purposes. The dissertation also demonstrates oligo-LacNAc synthesis in a cost-effective way. Normally, oligo-LacNAcs exist as an inseparable mixtures isomer in nature. By using our newly developed enzymatic system, defined lengths of oligo-LacNAcs were synthesized in a one-pot fashion by employing expressed NmGalT and HpGnT in the presence of UDP-Gal and UDP-GlcNAc. Also, we have demonstrated the versatility of the method by incorporating structurally more complex sialic acid residues with different linkages at the hitherto unknown internal Gal unit of oligo-LacNAc backbone in combination with α-2,3-sialyltransferase and α-2,6-sialyltransferase. Thus, we have achieved the synthesis of sialyl-oligo-LacNAcs; a hexa-saccharide with two repeating sialyl-LacNAc unit (4-26-2) and a nona-saccharide with three repeating sialyl-LacNAc unit (6-26-3), the attachment of which at the internal galactose unit was otherwise difficult by chemical means. With the enzymatic system, we can efficiently and quickly produce oligo-LacNAc derivatives. Finally to gain insights into the structure-activity studies, we have determined nine crystal structures of RmlA complexed with NDP-sugars, which we have synthesized enzymatically. Therefore, with the analysis of these structures, we can create amino acid mutation to improve the substrate tolerance and the catalytic efficiency of RmlA for accelerating progress in glycobiology.

Glycans are macromolecules that contain several classes. Glycans can play an important role in biological activities. Studying the cell surface glycans can provide a very powerful way to understand the fundamental process. Also it could help to regulate expected cell response. Thus it is very necessary to have a method to detect cell- surface glycans efficiently. An efficient method for glycan detection is necessary. Metabolic glycan labeling and chemoenzymatic glycan labeling are most commonly used. Chemoenzymatic glycan labeling is a rapid and sensitive method which also has high specificity. This method can be applied in both vitro and vivo. However the availability of unnatural sugar nucleotides functioned by bioorthogonal groups is the main limitation for chemoenzymatic labeling. In this thesis, UDP-GlcNAc and UDP-GalNAc derivatives were prepared for further chemoenzymatic labeling by using chemoenzymatic synthesis method.

博士
國立清華大學
分子醫學研究所
100
This thesis consists of 3 chapters. The first 2 chapters describe the molecular evolution of prokaryotic exceptionally large-sized genes (ELSGs) and nucleotide-sugar pyrophosphorylases/phosphorylases (NDP-sugar PPases/Pases). The third chapter describes a computer-aided drug design for searching bacterial UDP-glucose pyrophosphorylase (UGPase) inhibitors. In chapter 1, we have noted that, in contrast to the average gene size of approximately 1 kb in bacteria, 3 genes > 13 kb were present in Vibrio vulnificus. The finding prompted us to investigate the prevalence, possible function, and origin of ELSGs (>10 kb) in prokaryotes. Forty-two ELSGs (0.03%) were identified after searching more than 170,000 genes in 46 bacterial and 11 archaeal species. Homology analysis of these ELSGs indicated that, in addition to encoding non-ribosomal peptide synthetic enzymes, many ELSGs likely encode membrane-anchored proteins. Dot-matrix plot analysis of these ELSGs indicated that domain-duplication contributed significantly to size expansion. Other size expansion mechanisms were direct gene fusion, recombination of different genes, and horizontal gene transfer. In summary, ELSGs are commonly present in prokaryotes, and the evolutionary processes that have contributed to the formation of ELSGs are relatively heterogeneous. NDP-sugar PPases/Pases play a central role in providing sugar donor for the formation of glycoconjugates. Despite each of the enzymes has unique substrate specificity, they share homologous protein sequences and are therefore difficult to annotate. Chapter 2 describes our effort to better categorize these enzymes. More than 2,500 NDP-sugar PPases/Pases sequences were collected from the KEGG in this study and 86 representative sequences were selected. Phylogenetic and domain analyses revealed that members of GDP-Man PPase had the most diverse protein sequences implying that this enzyme is evolutionally closer to the common ancestor of the NDP-sugar PPases/Pases than other members of the family. Most NDP-sugar PPases/Pases were apparently derived by gene duplication although horizontal gene transfer, as in the case of eukaryotic UDP-Glc PPase, also contributed to the gene diversification. An evolutionary model for this group of enzymes was established by combining phylogenetic analysis and domain profiling. The core domains of each of the enzymes, trend of domain gain and loss, and evolutionary transition were demonstrated. These non-redundant 86 representative sequences may be used as the reference sequences for NDP-sugar PPases/Pases categorization. The low levels of sequence homology between prokaryotic and eukaryotic UGPase makes the enzyme a good target for antimicrobial drug development. Chapter 3 describes 22 UDP-Glc analogs and 97 candidate compounds, respectively selected from the ZINC and NCI compound databases using the protein docking program, LigandFit, that have potential to inhibit the bacterial UGPase activity. Five of the 97 candidate compounds were also identified by another docking program, Libdock. These 27 compounds represent potential bacterial UGPase inhibitors. The research interpreted the evolution of prokaryotic ELSGs, the categorization and evolutionary history of NDP-sugar PPases/Pases, and obtained the potential bacterial UGPases candidate inhibitors. The next step, we hope to obtain or synthesis these candidate compounds, or to screen the approved drugs in drug product database (new uses for old drugs) to perform the antimicrobial assay for the research and development of novel antimicrobial agents.